I nsect Molecular Biology (1992) 1(1), 25-36
Phylogeny of cytoplasmic incompatibility microorganisms in the parasitoid wasp genus Nasonia
(Hymenoptera: Pteromalidae) based on 16S ribosomal
DNA sequences
J. A. J. Breeuwer,' R. Stouthamer,' S. M. Barns,'
D. A. Pelletier, 3 W. G. Weisburg3 and J. H. Werren'
' Department of Biology, University of Rochester,
Rochester, NY 14627, USA
2
Department of Biology, Indiana University, Bloomington,
IN 47405, USA
3
GENE- TRAK Systems, Framingham, MA 01701, USA
Abstract
Cytoplasmic incompatibility results in embryo mortality in diploids, or all male offspring in haplodiploids,
when individuals carrying different cytoplasmic factors are crossed. Cytoplasmic factors have been
identified as intracellular micro-organisms. Microbei nduced cytoplasmic incompatibility is found in many
i nsect taxa and may play a role in reproductive isolation between populations. Such micro-organisms
cause bidirectional incompatibility between species of
the parasitoid wasp genus Nasonia. The phylogenetic
relationship of cytoplasmic incompatibility microorganisms (CIM) of different Nasonia species was
analysed using their 16S ribosomal DNA (rDNA) sequence. Two 16S rDNA operons were detected in the
CIM of each Nasonia species. Sequence analysis indicates that the Nasonia CIM are closely related and
belong to the alpha group of the Proteobacteria.
Keywords: incompatibility, micro-organisms, Nasonia, phylogeny.
Introduction
Cytoplasmic incompatibility (CI) results in embryo mortality
(Laven, 1957; Kellen etaL, 1981; Trpis etaL, 1981; Hsiao &
Hsiao, 1985; Wade & Stevens, 1985; Hoffmann et al.,
1986; Hoffman, 1988) or production of all male offspring
(Saul, 1961; Richardson et al., 1987; Breeuwer & Werren,
1990) i n crosses between individuals carrying different
cytoplasmic factors. Cytological observations in several
species revealed that eggs produced in incompatible
Received 10 December 1991; Accepted 18 March 1992. Correspondence:
J.A.J. Breeuwer.
crosses are normally fertilized, but syngamy of maternal
and paternal pronuclei is aborted, resulting in haploid
embryos (Jost, 1970; Ryan & Saul, 1968; Breeuwer &
Werren, 1990). I n diploid species these embryos are inviable. However, in haplodiploid organisms such as Nasonia, haploid embryos develop into males.
I n most cases cytoplasmic factors have been identified
as maternally inherited micro-organisms. Evidence comes
from cytological observations of micro-organisms in reproductive tissues (Yen & Barr, 1973; Kellen et al., 1981; Trpis
et al., 1981; Hsiao & Hsiao, 1985; Binnington & Hoffmann,
1989; O'Neill, 1989; Breeuwer & Werren, 1990), or mani pulation of compatibility through antibiotic treatment of
i nfected individuals (Yen & Barr, 1973; Kellen et al., 1981;
Trpis et al., 1981; Wade & Stevens, 1985; Hoffmann et al.,
1986; Hoffmann, 1988; Richardson et al., 1987; Breeuwer
& Werren, 1990; Montchamp-Moreau et al., 1991).
Micro-organisms that cause incompatibility in crosses
were first described in the Culex pipiens complex and
named Wolbachia pipientis (Hertig & Wolbach, 1936; Yen
& Barr, 1971). I n other systems of cytoplasmic incompatibility, the micro-organisms have also been classified as
Wolbachia sp. (Wright & Barr, 1980; Kellen et al., 1981;
Hsiao & Hsiao, 1985), although there was li ttle data to
support a monophyletic origin of these bacteria. Krieg &
Holt (1984) described Wolbachia as a miscellaneous group
of small, Gram-negative rods which are associated with
i nvertebrates. Because they cannot be cultured easily
outside the host tissue, further classification of these microorganisms, using traditional biochemical classification
methods, is difficult. Moreover, these biochemical characters do not necessarily correlate with phylogenetic relatedness. With recent advances in molecular biology, such as
the polymerase chain reaction (PCR: Saiki etaL,1988), it is
now possible to identify fastidious micro-organisms using
DNA sequence information. In particular the 16S rDNA is
useful because of its relatively small size and conserved
function (Fox et al., 1980; Woese, 1987). Ribosomal DNA
sequence data from several members of the order Rickettsiales have already demonstrated extensive phylogenetic
divergence between genera of this taxon (Weisburg et al.,
1989;1991).
Cytoplasmic incompatibility i s widespread among
25
26
J. A. J. Breeuwer et al.
i nsects, e.g. Lepidoptera (Kellen et al., 1981), Diptera
(Wright & Barr, 1980; Yen & Barr, 1973; Hoffmann, 1988;
Hoffmann et al., 1986), Hymenoptera (Richardson et aL,
1984), and E. risticii is the causative agent of equine
monocyte ehrlichiosis (Holland et al., 1985). Both are
i ntracellular, in contrast to Rickettsia which reside i n
1987; Breeuwer & Werren, 1990), Coleoptera (Wade &
Stevens, 1985; Hsiao & Hsiao, 1985). This may indicate
that cytoplasmic incompatibility micro-organisms (CIM) have
been present i n insects for a long time, and have diverged
i n association with their host species. A second possibility
is that CIM have been laterally transferred between insect
taxa. The third possibility is that CIM have evolved many
ti mes i ndependently from parasitic or symbiotic bacteria
associated with insect hosts. To distinguish between these
possibilities, we need to clarify the phylogenetic relationships between CIM from different host species.
The parasitoid wasp genus Nasonia contains three
closely related species in North America (Darling & Werren, 1989). Natural populations of each Nasonia species
are infected with micro-organisms that cause bidirectional
i ncompatibility between their host species, resulting in the
production of all male offspring (Breeuwer & Werren,
1991). I nfected individuals from the same wasp species
are compatible, whereas those from different wasp species
are i ncompatible. The elimination of micro-organisms restores compatibility, and hybrid offspring are produced.
These observations suggest that each wasp species harbours its own strain of micro-organism.
Here we present the sequences of the 16S rRNA gene of
CIM from each Nasonia species. These sequences are
used to clarify their phylogenetic relationships and identify
their closest known relatives.
vacuoles (Krieg & Holt, 1984). Traditionally, CIM, such as
those found i n the mosquito C. pipiens (Yen & Barr, 1971),
the almond moth Ephestia cautella (Kellen et al., 1981),
and alfalfa weevil, Hypera postica, (Hsiao & Hsiao, 1985),
were placed in the genus Wolbachia together with W.
persica. However, according to Weisburg et al. (1991),
based on 16S sequences, W. persica does not belong to
the alpha group but rather to the gamma group of Proteobacteria. I n contrast, we have found that the Nasonia CIM
do fall in the alpha group. Similarly, O'Neill etaL (1992) has
also found that the 16S sequences of the CIM of several
other insects (C. pipiens, E. cautella, D. simulans, Aedes
albopictus, Tribolium confusum and H. postica) place them
i n the alpha group between Ehrlichia and Anaplasma.
Sequence similarity between complete 16S rDNA sequences of CIM from Nasonia species is high, about 95%.
For finer scale analysis of their relationships, we considered those positions which showed variation between
Nasonia CIM clones. Based upon these sites, the sequences can be roughly divided into two groups, which are
50-90% homologous within and 30% homologous between groups (Table 2).
Surprisingly, different CIM 16S rDNA sequences from
the same wasp species can be more different from one
another than from homologous sequences of the other
species (Fig. 3). Additional sequencing of clones confirms
the presence of two ribosomal types (U. Bergthorsson,
J.A.J. Breeuwer and J.H. Werren, unpublished results).
There are at least three explanations for the pattern of
sequence variation within PCR amplification from single
Nasonia strains, (i) variation is a PCR or sequencing
artifact, (ii) there are two strains of CIM, each with one
ribosomal operon, or (iii) there i s one CIM with at least two
ribosomal operons. If the observed clonal sequence variation is a result of polymerase errors during PCR amplification or sequencing, variable nucleotide positions are
expected to be distributed randomly among sequences.
This is not the case.
The evidence i s strong that the two sequence subgroups
Results and discussion
DNA fragments of 1.5 kb were recovered from ovaries of
CIM-infected wasp strains after polymerase chain reaction
(PCR) amplification with prokaryote rDNA-specific
primers. In contrast, no PCR product was recovered from
asymbiont wasp strains. Two clones were sequenced
entirely from each wasp species (Fig. 1). Each CIM 16S
rDNA sequence showed a 25 nucleotide deletion between
E. coli basepair positions 455 and 480 (Brosius et al.,
1978; Woese et al., 1983). Two additional deleted helices
between basepair positions 180 and 220 were also present, and place CIM in the alpha subdivision of Proteobacteria (Woese, 1987; Woese et al., 1983; Stackebrandt
et al., 1988).
Comparison of the N. vitripennis CIM 16S rDNA sequence with other bacterial sequences shows that they are
related to the Rickettsiales i n the alpha subdivision (Weisburg et al., 1991). Their closest known relatives, based
upon similarity data (Table 1), are Anaplasma marginale
and Ehrlichia risticii (Weisburg et al., 1991). Anaplasma
marginale is an obligate intracellular parasite of erythrocytes in ruminants, transmitted by arthropods (Krieg & Holt,
represent real (functional) ribosomal genes rather than
artifacts. First, 22 sites were found to be diagnostic for the
two subgroups, i.e. each subgroup was characterized by
one of the two nucleotide variants (see Table 3) present.
Ten of these sites occurred in unpaired regions of the
ribosomal gene (e.g. stemloops), where sequence variations are more easily tolerated. Of the 12 sites occurring in
paired regions (stems), four were actually compensatory
changes affecting mutually paired sites. Six involved GU
pairing, which is commonly found in the secondary structure of 16S rRNA (Woese et al., 1983), and these noncanonical pairs do not necessarily impair ribosomal func-
Phylogeny of Nasonia incompatibility microbes
27
1 50
I
I
I
I
I
CONS=90t;
GAACGCTGGCGG - GC T A ACATGCAAGTCG CG
ATTGAACGCTGGCGGCA-GGCCTAACACATGCAAGTCGAACGGTAACAGGAAGAAGCTTG
E. coli
A. margina AACGAACGCTGGCGGCA-AGCTTAACACATGCAAGTCGAACGGACCGTATACGCAGCTTG
El
V12
G6
L36
AATGAACGCTGGCNGCA-GGCCTAACACATGCAAGTCGAACGGAGTTATATTGTAGCTTG
. ............ G..............................................
. . ........................ G..C.........
. ............ G..............................................
V27
G17
L44
. . . .......... G..............................................
. ........ C...G..................... C........................
1 100
1
1
1
1
1
T
A T CC
G GGC ACGGGTG GTAA
G
CONS=90$
CTTCTTTGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGG
E. coli
A. margina CTGCGTGTATGGTTAGTGGCAGACGGGTGAGTAATGCATAGGAATCTACCTAGTAGTATG
El
V12
G6
L36
CTATGGTATAACTTAGTGGCAGACGGGTGAGTAATGTATAGGAATCTACCTAGTAGTACG
............................................................
. . ............ G.............................................
. ...........................................................
V27
G17
L44
1 150
1 200
1
1
1
1
GAAA
TAATACC AT
AAAG
CONS=90%
G A A C
GGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGAC
E. coli
A. margina GGATAGCCACTAGAAATGGTGGGTAATACTGTATAATCCTGC-GGGGGAAAGA------El
V12
G6
L36
GAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAAA------. .........................................NN................
................................... GT..... NN................
..........................................TN................
V27
G17
L44
. . ........................................NN................
..........................................NN................
1
GA 1
1 AT AG TGTTGG
GGTAA GG
250CONS=9%
CTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGG
E. coli
A. margina -TTTA------ TCGCTATTAGATGAGCCTATGTCAGATTAGCTAG'1'TGG'EG000TAATGG
1
El
V12
G6
L36
1
-TTTA------ TTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGAGTAATAG
. . ..........................................................
. . ..........................................................
. ...........................................................
V27
G17
L44
1 300
1
I
1
1
I
TA C G CTGAGAGG GA C G CACA TGG ACTGAG
CONS=90$
C ACCAAG C A GA
CTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAG
E. coli
A. margina CCTACCAAGGCGGTGATCTGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAG
El
V12
G6
L36
CCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAG
. ...........................................................
. ...........................................................
. ...........................................................
V27
G17
L44
Figure 1. 16S rRNA sequence alignment of E. coli, A. marginale, and, CIM from Nasonia vitripennis (El, V12, V27), N. girauld (G6, G17), and
N. longicornis (1-36, 1-44). The eubacterial consensus sequence shows regions that are more than 90% conserved in approximately 85 bacterial sequences
(from Weisburg et al., 1991). Position numbering is based upon E coli (Brosius et al., 1978). El is used as a concensus. In other CIM sequences sites
identical to El are replaced by a dot and only variable sites are listed. The nucleotide sequence data reported in this paper will appear in the EMBL,
GenBank and DDBJ Nucleotide Sequence Databases under the following accession numbers: E1 = M84686, V12 = M84687, V27 = M84688,
G6 = M84689, G17 = M84690, L36 = M84691, L44 = M84692. (N = G or A or T or C; - = deletion.) (Continued).
28
J. A. J. Breeuwer et al.
350
CONS=90%
ACACGG CCA ACTCCTACGGGAGGCAGCAGT I GGAAT TT I CAATGG G AA C
E. coli
ACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCC
A. margina ACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCC
El
V12
G6
L36
ATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCC
.........................................................NNN
.........................................................NNN
.........................................................NNN
V27
G17
L44
. ........................................................NNN
. ........................................................NNN
. ........................................................NNN
400
I
CONS=90%
TGA I
AGC A GCCGCGTG
GA GA G
T GG
GTAAA
CT T I
c. coli
TGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGG
A. margina TGATCCAGCTATGCCGCGTGAGTGAGGAAGGCCT'V'AGGG'TT'GTAAAACTCTTTCAGTAGG
El
V12
G6
L36
TGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAG
. ...........................................................
. ................................. A.............. G..........
. ...........................................................
V27
G17
L44
1 450
1
500
1
1
1
CONS=90%
GA G
TGAC TA
A AAGC CGG
E. coli
GAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGG
A. margina GAAG-------------- ATAA----------- TGACGGTACCTACAGAAGAAGTCCCGG
El
V12
G6
L36
GAAG-------------- ATAA----------- TGACGGTACTCACAGAAGAAGTCCTGG
. . . . . .......................................................
.................. ...G......................................
............................................................
V27
G17
L44
...T........................................................
550
CONS=90%
CTAACT GTGCCAGCAGCCGCGGTAATAC AGG GC AGCGTT
CGGA T A TG
E. coli
CTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTG
A. margina CAAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGGGCAAGCGTTGTTCGGAATTATTG
El
V12
G6
L36
CTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTG
............................................................
............................................................
............................................................
V27
G17
L44
1 600
CONS=90%
GGCGTAAAG G
G AGG GG
AGT G GT AAA I
GCT AAC
E. coli
GGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTG
A. margina GGCGTAAAGGGCATGTAGGCGGTTTGGTAAG7"i'AAAGGTGAAATACCAGGGCTTAACCCT
El
V12
G6
L36
GGCGTAAAGGGCGCGTAGGCTGGTTAATAAGTTAAAAGTGAAATCCCGAGGCTTAACCTT
. ................. NN-.A...G.................... A..... C......
. ................. NN..A...G.................... A..... C......
. ................. NN..A.....................................
V27
G17
L44
.................. NN-.A...G.................... A..... C......
. ................. NN-.A...G.................... A..... C......
.................. NN-.A...G.................... A..... C......
Figure 1. Continued.
Phylogeny of Nasonia incompatibility microbes
1 650
I
I
1
I
I
CONS=90%
A AC
CT GA
AG GG
GAATT
GTGT
E. coli
GGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGT
A. margina GGGGCTGCTTTTAATACTGCAGGACTAGAGTCCGGAAGAGGATAGCGGAATTCCTAGTGT
El
V12
G6
L36
GGAATTGCTTTTAAAACTATTAATCTAGAGATTGAAAGAGGATAGAGGAATTCCTGATGT
..... A............ GC............... G................... AG...
.................. G.................................... AG...
............................................................
V27
G17
L44
. ................. GC............... G................... AG...
. . ................ G.................................... Ad...
. ................. GC................................... AG ...
1 700
1
1
1
I
1
CONS=90%
AG GGTGAAAT CGTAGA AT
A GAA ACC T GCGAAGGC
CTGG
E. coli
AGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGA
A. margina AGAGGTGAAATTCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTGTCTGGTCCG
El
V12
G6
L36
AGAGGTAAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCA
...... G.....................................................
. ...........................................................
. ...........................................................
V27
G17
L44
. ..... G.....................................................
. ..... G.....................................................
. ..... G.....................................................
1 750
1
1
1
1 800
1
CONS=90%
A TGAC CT A G CGAAAGCGTGGGGAGC AACAGGATTAGATACCCTGGTAGTCC
E_ -Ii
AGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCC
A. margina GTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTACTCC
El
V12
G6
L36
AATCTGACGCTGAAGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCC
. . . .......... G..............................................
. . ..........................................................
. ..................... N.....................................
V27
G17
L44
. ............ G..............................................
. . ........... G..............................................
. ............ G..............................................
1 850
1
I
I
1
1
CONS=90%
ACGC TAAACGATG
GT G
G
T
AG AAC
E. coli
ACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAAC
A. margina ACGCTGTAAACGATGAGTGCTGAATGTGGGGGC-TTTT--GCCTCTGTGTTGTAGCTAAC
El
V12
G6
L36
ACGCTGTAAACGATGAATGTTAAATATGGGAAG-TTT--ACTTTCTGTATTACAGCTAAC
..................................... T......................
. . . .........................................................
V27
G17
L44
. . ................................... T......................
. .................................... T......................
. .................................... T......................
1 900
1
1
I
1
1
CONS=90%
GC TAA
CCGCCTGGG AGTACG CGCAAG T AAACTCAAA GAATTGACGG
E. coli
GCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGG
A. margina GCGTTAAGCACTCCGCCTGGGGACTACGGTCGCAAGACTAAAACTCAAAGGAATTGACGG
El
V12
G6
L36
GCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGG
........................................ G...................
............................................................
............................................................
V27
G17
L44
Figure 1. Continued.
29
30
J. A. J. Breeuwer et al.
1 950
I
I
I
I
1
CONS=90%
GG -000CACAAGCGG GGAG ATGTGGTTTAATTCGA G ACGCG
GAACCTTACC
F, coli
GGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAACA.4CCTTACCT
A. margina GGACNCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCA
El
V12
G6
L36
GGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCA
. ...........................................................
. ...........................................................
............................................................
V27
G17
L44
I
I
CONS=90$
I TTGACAT
1000 -GA AI
E. coli
GGTCTTGACATCCACGGAA-GTTTTCA-GAGATGAGAAT-GTGCCTTCG-GGAACCGTGA
A. margina CTTCTTGACATGGAGGCTAGATCCTTCTTAACAGAAGGGCG-CAGTTCGGCTGGGCCTCG
El
V12
G6
L36
CTTCTTGACATGGAAATCATACCTATTCGAAGGGATAGG-GTCGGTTCGGCCGGATTTTA
. . C.........................................................
. . C....................................................... C.
. . C....................................................... C.
V27
G17
L44
. . C................................................... G...C.
. . C................................................... G...C.
. . C.................................................... G...C.
1 1050
1
I ll
1
1
1
CONS=90%
ACAGGTG TGCATGG TGTCGTCAGCTCGTG C.-A. TGTTGGGTTAAGTCCCGCAA
E. coli
GACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAA
A. margina CACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA
El
V12
G6
L36
CACAAGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA
.............................................................
............................................................
............................................................
V27
G17
L44
.... G.......................................................
.... G.......................................................
. ... G.......................................................
00
1 1150
1
CONS=90%
CGAGCGCAACCC
GTT C A C
G G- ACTC
ACTG
°_. ^_oli
CGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTC-CGGCCGGG-AACTCAAAGGAGACTG
A. margina CGAGCGCAACCCTCATCCTTAGTTACCAGCGGGI'AAT'VCCGGG-CACTTTAAGGAAACTG
El
V12
G6
L36
CGAGCGCAACCCTCATCCTTAGTTGCTATCAGGTAATGCTGAGT-ACTTTAAGGAAACTG
. ...........................................................
. ...........................................................
. ........................................... G...............
V27
G17
L44
. ....................... A.C.............. G.-G...............
. ....................... A.C.............. G.-G...............
. ....................... A.C.............. G.-G...............
1 1200
I
I
CONS=90%
CC
G AA
GGAGGAAGG G GGA GACGTCAA TC TCATG CCCTTA G
G
E. coli
CCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAG
A. margina CCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCAGCACGGCCCTTATGGGGTG
El
V12
G6
L36
CCAGTGATAAGCTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCTTTATGGAGTG
. ..................................................... A.....
. ...........................................................
V27
G17
L44
. ......... A...................................... C..........
.......... A...................................... C..........
.......... A...................................... C..........
Figure 1. Continued.
Phylogeny of Nasonia incompatibility microbes
1 1250
I
1
1
I
CONS=90%
GGCTACACACGT CTACAATGG
ACA G G GC A
G GA
AGC A
E coli
GGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCT-CGCGAGAGCAAGCGG
A. . margin. GGCTACACACGTGCTACAATGGCGACTACAATAGGTTGCAACGT-CGCAAGGCTGAGCTA
El
V12
GGCTACACACGTGCTACAATGGTGTCTACAATGGGCTGCAAGGTGCGCAAGCCTAAGCTA
. ...........................................................
................................... T...................... C.
................................... T........................
G6 L36
........................ G................ A...... G..G...... C.
........................ G................ A...... G..G........
........................ G................ A...... G..G........
V27 G17 L44
1
1300
I
1
1
A C
CTGCAACTCG
C TGAAG
GGA
AAA
TC AGT CGGAT G
ACCTCATAAAGTGCGTCG1'AGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGA
margina ATCCG-TAAAAGTCGTCTCAGTTCGGATTGTCC'1'C'1'G'1'AACTCGA000CATGAAGTCGGA
CONS=90°s
E, cnli
A.
El
V12
G6
L36
ATCCC-TAAAAGACATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTTGGA
. . . . . . . . . . . . . . . . . . . . . . ......................................
. . . . ........................................................
. . . . . . . . . . . . . . . . . . ..........................................
V27
G17
L44
. . ... T...... C.................................. G............
. .... T...... C.................................. G............
. .... T...... C.................................. G............
1 1350
1
1
1
CGGTGAATACGTTC CGGG CTTGTACACAC
ATCAG A
E. coli
ATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACAC
A. margina ATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACACAC
1
CONS=90%
T GCTAGTAATCG
El
V12
G6
L36
ATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACACAC
. . . . . .......................................................
............................................................
. . ..........................................................
V27
G17
L44
. ....................... C...................................
........................ C...................................
........................ C...................................
1 1400
1
1 1450
1
1
1
CGCCCGTCA
CA G AG
AAG
AACC
GGA
E. coli
CGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGG
A. margina TGCCCGTCACGCCATGGGAATTGGCTTAACTCGAAGCTGGTGCGCCAACCGTAAGGAGGC
CONS=90$
El
V12
G6
L36
TGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTAATGGCCTAACCGCAAGGAAGG
. . . . ...................................................-....
. . .................. .C......................................
. ...........................................................
V27
G17
L44
. ....................................... GCA.............. G..
. ....................................... GCA............ -.G..
. ....................................... GCA.............. G..
C A
GT G
A TGGG
CGCTTACCACTTTGTGATTCATGACTGGGGTG
A. margina AGCCATTTAAGGTTGGGTCGGTGACTGGGGTG
CONS=90%
R. coli
El
V12
G6
L36
AGTTATTTAAAGTGGGATCAGTGACTGGGGTG
. . . ...................... C......
. . ..............................
. ...............................
V27
G17
L44
. . . . . . . . ........... G............
. . ................. G........ A...
. .................. G............
Figure 1. Continued.
31
32
J. A. J. Breeuwer et al.
Escherichia coli
Serratia marcescens
Gamma subdivision
of Proteobacteria
Proteus vulgaris
Arsenophonus nasoniae (sonkiller)
Wolbachia persica
Rickettsia prowazeki
Rickettsia rickettsi
Alpha subdivision
of Proteobacterio
Evolutionary distance
Figure 2. Phylogenetic tree with a Nasonia vitripennis CIM (El), based on evolutionary distance (Olsen, 1988). The Proteobacteria are divided into four
groups. Members of two groups, alpha and gamma, are shown. E. coli (gamma) is used as an outgroup to root the tree.
Table 1. Percentage sequence similarity, above diagonal, and evolutionary distance (x 100), below diagonal, for Nasonia CIM ( E1) and 16 other bacteria
( Weisburg et al., 1991) belonging to the alpha and gamma subdivision of the Proteobacteria
Bacterium
Ecol
Escherichia coli
Serratia marcescens
Proteus vulgaris
Arsenophonus nasoniae
Wolbachia persica
Rickettsia prowazekii
Rickettsia rickettsii
Ehrlichia risticu
Nasonia CIM (El)
Anaplasma marginale
Rhodopseudomonas palustris
Brucella abortus
Rochalimaea quintana
Agrobacterium tumefaciens
Rhodospirillum molischiarum
Rhodospirillum rubrum
Rhodospirillum fulvum
2.5
4.8
6.6
14.4
19.9
20.1
23.1
20.5
19.7
19.7
17.9
18.3
18.5
17.4
16.1
17.4
S.mar Rvul A.nas
caper R.pro R.ric
97.8
3.8
6.5
14.3
19.5
19.7
22.1
19.9
19.7
20.7
17.9
18.3
18.4
17.9
16.8
17.9
86.9
87.0
86.5
85.2
20.1
20.3
23.3
22.4
20.9
18.9
19.3
18.9
17.7
17.5
17.4
17.5
95.3
96.2
8.2
17.9
19.6
19.9
23.5
21.2
20.7
21.0
18.5
18.2
17.9
17.9
17.1
17.9
93.7
93.8
95.0
16.5
19.0
19.2
24.4
21.7
21.0
22.0
18.7
19.0
18.5
18.5
18.0
18.5
82.5
82.8
82.7
83.2
82.4
9.3
15.6
15.5
13.9
14.1
12.8
13.0
13.5
13.5
14.9
13.8
82.3
82.7
82.6
83.0
82.2
99.1
15.3
15.3
13.8
14.3
12.6
12.6
13.3
13.5
15.0
13.6
Eris
CIM
80.1
80.9
79.8
79.2
80.0
85.9
86.1
14.2
14.1
17.6
16.0
17.3
15.8
17.7
17.8
18.0
82.0
82.5
81.6
81.2
80.6
86.0
86.1
87.1
11.2
18.1
16.6
16.4
16.0
17.5
16.7
17.9
A.mar R.pal B.abo R.qui A.tum R.mol R.rub RJul
82.7
82.7
81.9
81.7
81.7
87.3
87.4
87.2
89.6
16.7
14.8
14.3
14.2
14.5
15.6
14.8
Clone
El
V12
G6
L36
V27
G17
L44
El
V12
G6
L36
V27
G17
L44
-
67
-
67
55
-
88
59
76
-
29
36
32
34
-
29
36
32
38
90
-
27
38
32
34
88
92
-
82.7
81.9
81.7
80.9
83.3
87.2
87.0
84.3
83.9
85.0
9.1
9.5
9.4
9.7
10.5
9.9
84.1
84.0
83.6
83.4
82.9
88.3
88.4
85.6
85.1
86.6
91.4
4.1
4.7
8.7
9.7
8.8
83.7
83.7
83.9
83.2
83.3
88.1
88.4
84.6
85.3
87.0
91.0
96.0
5.0
8.2
10.6
8.4
83.6
83.6
84.1
83.6
84.2
87.6
87.8
85.8
85.6
87.0
91.2
95.5
95.2
7.9
10.1
8.4
84.5
84.1
84.1
83.6
84.4
87.7
87.6
84.3
84.4
86.8
90.9
91.8
92.2
92.5
7.9
3.6
85.5 84.5
85.0 84.1
84.7 84.0
84.0 83.6
84.5 84.4
86.5 87.4
86.4 87.5
84.2 87.5
85.1 84.0
85.9 86.6
90.2 90.7
90.9 91.7
90.1 92.6
90.5 92.1
92.5 99.6
- 92.3
8.1
-
Table 2. Percentage similarity (Olsen, 1988)
solely based on variable nucleotide sites (59, gaps
included) for the shortest common sequence,
G17. Weight given to gaps is 1. (V and E = N.
vitripennis, G = N. giraulti, L = N. longicomis.)
Phylogeny of Nasonia incompatibility microbes
8
6
EI
r2- V27
1
-3- G17
I - L44
Number of nucleotide substitutions
Figure 3. Most parsimonious phylogenetic tree of Nasonia CIM using
branch-and-bound method of PAUP (Swofford, 1990). Only variable sites
for the shortest common sequence, G17 (52 sites) were included.
A. marginale was used as an outgroup to root the tree. (V and E _
N. vitripennis, G = N. giraulti, L = N. longicomis.)
tion. Only two changes disrupted correct pairing. Both of
these occurred in a region (1440-1442) already experienci ng disruption of perfect base pairing (Woese et al., 1983).
The finding that nucleotide differences at diagnostic sites
are consistent with 16S rRNA secondary structure shows
that variation at these sites is not an artifact but is indicative
of the presence of more than one ribosomal operon.
It is currently unknown whether CIM have more than one
ribosomal operon. Thus, each operon may represent two
different bacterial strains present in each wasp species.
Alternatively, each Nasonia species may harbour a single
CIM strain with two ribosomal operons. Because little is
known about the amount of sequence variation between
ribosomal operons within bacterial strains, it is not possible
to distinguish between these two possibilities solely on the
bases of sequence information. Prokaryotes typically have
a few (e.g. 1-10) ribosomal operons (Pace et al., 1986) but
almost all have more than one. Only a few studies have
examined sequence variation in ribosomal operons within
unicellular species. The mitochondrial genome of Tetrahymena contains two genes encoding the large subunit rRNA
that differ at 5 of 2595 positions (Heinonin et al., 1990).
Mylvaganam & Dennis (1992) observed 74 differences in
1472 positions of two 16S rRNA genes from the archaebacterium, Haloarcula marismortui, and both types are
present in functional ribosomes. In contrast, Dryden &
Kaplan (1990) did not find any difference between 16S
rDNA of three ribosomal RNA operons of Rhodobacter
sphaeroides.
I n principle, one nucleotide difference is sufficient to
distinguish between species or strains as long as it is a
consistent difference. However, to determine this, multiple
sequences are needed from different strains of each
species. In addition, greater confidence is obtained when
phylogenetic trees are based on more differentiating
33
characters. Therefore, sequence information on more
rapidly evolving genes or ribosomal spacer regions, e.g.
between 16S and 23S rDNA, is necessary to resolve the
phylogenetic relationships among CIM.
I n addition, it is useful to obtain the complete 16S rDNA
sequence because variable positions can be restricted to
particular regions of the 16S gene (Mylvaganam & Dennis,
1992). In Nasonia CIM diagnostic sites are restricted to a
region between nucleotide position 1000 and 1500, and
most changes are associated with stem loops.
Taq polymerase or sequencing errors may be partly
responsible for non-diagnostic nucleotide differences between the two ribosomal types. Sequence information of
multiple clones of each type are needed to determine
possible Taq or sequencing errors.
All CIM appear to be closely related and group into
a monophyletic assemblage of intracellular parasites
( O'Neill et aL, 1992). We cannot yet identify potential
ancestors. Because CIM occur in diverse insect taxa, this
suggests lateral transmission of the micro-organisms between insect groups. One possibility is transfer between
i nsects that are associated with each other. For instance, in
the case of Nasonia, CIM-like 16S rDNA sequences have
also been found in Protocalliphorid flies, which Nasonia
wasps parasitize (J.A.J. Breeuwer and J.H. Werren,
unpublished results). Associations such as these are likely
avenues of intertaxon transfer.
Experimental procedures
Wasp strains
Total genomic DNA was isolated from the following wasp strains.
Labll, l aboratory strain Leiden, The Netherlands,
Europe. Asymc, strain of Labll cured of CIMs in 1986.
N. vitripennis.
RV2, collected in Virginia, USA, 1986. RV2T, strain of
RV2 cured of CIMs in 1988.
N. giraulti.
N. longicomis. IV7, collected
of I V7 cured of CIM in 1989.
in Montana, USA, 1986.
I V7T
strain
As indicated, symbiont free lines were derived from the wild-type
strain of each species by feeding females tetracycline (1 mg ml - '
tetracycline in 10% sucrose) prior to egg laying. This was repeated
for three generations, at which time no bacteria could be detected
in the cytoplasm of freshly laid eggs stained with 2% lacmoid
(Breeuwer & Werren, 1990). These strains have been maintained
free of CIM for several years of laboratory maintenance and show
altered compatibility relationships in intra- and interspecific
crosses (Breeuwer & Werren, 1990).
DNA preparation
DNA was prepared from 5 to 10 ovaries (all strains) or 500 to 1000
eggs (only Labll and Asymc). Ovaries were dissected in Tris buffer
34
J. A. J. Breeuwer et al.
Table 3. Variable positions between sequenced Nasonia CIM clones and their location inferred from the 16S rRNA secondary structure of E. coli
(Woese, et al., 1983). Diagnostic positions are in boxes. (V and E = N. vitripennis, G = N. giraulti, L = N. longicornis.)
Clone
Position
El
V12
G6
L36
V27
37
62
74
77
101
182
183
421
436
450
468
587
589
593
614
620
632
645
646
662
682
683
693
760
844
907
989
1037
1041
1047
1127
1129
1143
1145
1146
1171
1210
1215
1245
1256
1262
1264a
1268
1271
1274
1278
1285
1292
1327
1364
1421
1440
1441
1442
1455
1457
1479
1485
1488
U
G
A
U
A
C
G
U
C
G
A
U
G
A
G
U
U
A
U
G
G
A
A
A
A
U
A
U
A
G
U
A
U
U
G
A
U
A
C
G
U
C
G
A
A
G
A
C
A
G
C
G
A
G
G
G
U
G
C
A
U
A
G
U
A
U
G
G
C
G
G
U
A
G
G
G
U
A
G
A
C
U
G
U
A
A
G
A
A
A
C
A
C
A
G
U
A
U
A
U
G
U
C
G
G
A
C
A
U
A
U
A
U
C
G
G
A
C
U
A
A
U
U
U
G
G
G
A
A
A
G
G17
L44
A
U
G
U
U
G
G
A
C
A
C
U
G
A
U
A
C
G
U
C
G
A
U
A
A
G
U
U
A
U
A
G
A
A
A
A
C
A
C
A
G
U
A
U
G
A
U
G
U
U
G
G
A
C
A
U
U
G
A
U
A
C
G
U
C
G
A
A
G
A
C
U
G
C
G
A
G
G
G
U
A
C
G
C
G
A
C
G
U
C
G
A
G
G
A
C
U
G
U
A
A
G
G
G
U
A
C
G
C
G
A
C
G
C
C
A
U
A
C
G
U
C
U
A
A
G
A
C
U
G
C
A
A
G
G
G
U
A
C
G
C
G
A
C
G
G
G
C
G
G
C
A
G
G
C
G
G
C
A
G
G
C
G
G
C
A
A
A
U
U
U
G
G
G
G
A
C
U
C
G
C
U
G
C
A
G
G
A
U
U
C
G
C
U
G
C
A
G
G
A
U
U
C
G
C
U
A
A
U
U
U
G
G
A
A
U
C
U
G
G
A
A
C
G
A
A
U
G
A
A
U
G
G
G
U
G
G
G
U
A
G
G
U
G
C
A
Position
paired
unpaired
paired
paired
unpaired
unpaired
unpaired
unpaired
paired
unpaired
unpaired
paired
paired
paired
paired
unpaired
paired
paired
paired
paired
paired
paired
unpaired
unpaired
unpaired
unpaired
paired
paired
paired
paired
unpaired
unpaired
unpaired
unpaired
unpaired
paired
paired
paired
paired
unpaired
unpaired
unpaired
unpaired
unpaired
unpaired
unpaired
unpaired
paired
paired
unpaired
paired
paired
paired
paired
unpaired
paired
paired
paired
paired
Nucleotide
547
A
96
92
A
G
406
G
754
650
646
626
C
U
UCUU CUC
U
606
594
593
744
708
707
G
U
AGGA GGG
U
U
U
1216
1003
909
1210
G
U
A
UC
1165
1047
990
1292
U
AG
C
AC
1245
1310
UG
U
1479
1461
1462
1463
AAAA GGG
G
U
U
1445
1421
1415
1412
U
UUCU UUU
G
C
Phylogeny of Nasonia incompatibility microbes
[10 mm Tris (pH 8.3), 2.5 mm MgC12 , 50 mm KCI] on a sterilized
slide. Samples were transferred into a 0.5 ml microfuge tube
containing 0.1 ml sterile zirconium beads (Biospec Product, Bartl esville, OK), 200#1 Tris, 10 ul 20% SDS and 200,ul phenol. The
order of tissue collecting from each Nasonia species was first
symbiont strain followed by the asymbiont strain of the same
species. This allowed a positive check for cross contamination.
Samples were shaken in a mini-Bead beater (BioSpec Products)
for 3 min. Phases were separated by 10 min spinning in a
microcentrifuge. The DNA was then ethanol precipitated and
resuspended in 20 ul sterile TE buffer [10 mm Tris (pH 8.0), 1 mm
EDTA]. All solutions were filter sterilized ((D 0.22um) before use to
reduce the risk of contamination.
35
et al., 1985) using conserved positions as guidelines. All positions
in which base composition was not at least 50% conserved were
eliminated (Weisburg et al., 1991). The percentage sequence
similarity for each pair of sequences was calculated after alignment as the percentage identical basepair positions of the total
number of common basepair positions (Olsen, 1988). The estimated evolutionary distance is based on the number of substitutions per sequence position (Jukes & Cantor, 1969).
Phylogenetic trees were constructed by the method of Olsen
(Olsen, 1988; Swofford & Olsen, 1990) based on pairwise evolutionary distance data and by the branch-and-bound method of
PAUP using actual sequence data (Swofford, 1990; Swofford &
Olsen, 1990).
DNA amplification and purification
Bacterial 16S rDNA was amplified using PCR in a volume of 50 ul
[1-2 ul DNA sample, 5 ul 10 x buffer (Promega), 1 ul nucleotide
mix (10 mm each), 0.625ul 20 mm primer 1, 0.625ul 20 man primer
2, 0.5 ul taq polymerase (Promega), and ddH20 was added to a
final volume of 50 ul]. Because PCR is a very sensitive technique
and can amplify DNA that is present at very low concentration,
extreme care was taken to avoid, but also detect, cross contamination between samples. Again all solutions were filter sterilized
( (D 0.22 um) before use to reduce the risk of contamination. Two
primer pairs; fD1 and rP1 for ovaries and fD2 and rP2 for eggs,
which specifically hybridize to bacterial 16S rDNA, were used
( Weisburg etaL, 1991).
The PCR reaction cocktail was prepared in one batch and then
added to each sample. The remainder of the cocktail was run
along as a control for contamination. PCR cycling conditions were
one cycle (1 min at 94°C, 2 min at 55°C, 3 min at 72°C), 28 cycles
(15 s 92°C, 1 min at 60°C, 3 min at 72°C), and 1 cycle (15 s at 92°C,
1 min at 60°C, 10 min at 72°C) (Ericomp thermal cycler). After
PCR, 1 ul of amplified product was run on a 1% agarose gel to
check for product. The remainder of the PCR product was purified
with the Geneclean kit (1310101 Inc., La Jolla, CA) and dissolved in
20ul ddH 2 0.
Cloning and sequencing
Egg PCR product was restriction digested with San and BamHI
and ligated into a similarly cut pGEM-4 vector (Promega). Ovarial
PCR product was directly cloned into T-tailed M13mp18 vector
(Marchuk et aL, 1990; Sambrook et al., 1989). Plasmid clones
were sequenced with Sequenase v 2.0 kit (US Biochemical Corp.)
according to their specifications and run on buffer gradient gels
(Biggin et al., 1983). Sequencing primers were 357f, 530f, 926f,
1114f (Lane, 1991), universal primer (5'GTTTT000AGTCACGAC), and 65f (5'GTGGCAGACGGGTGAGT). Egg PCR product
(E1) was sequenced in both directions, whereas ovarial PCR
products (V12, V27, G6, G17, L36, L44) were only sequenced
from 5'--> 3'. Because Nasonia CIMs may be closely related, PCR
or sequencing errors could obscure phylogenetic analysis. Therefore two clones of each species were sequenced completely to
detect microheterogeneity.
Estimation of evolutionary distance and phylogenetic tree
The 16S rDNA sequences of CIM were manually aligned with the
accepted secondary structure of E. coli (Woese etaL, 1983; Gutell
Acknowledgements
We would like to thank Bill Burke for technical advice, Thomas
Eickbush for use of facilities, Lasse Lindahl and Jan Zengel for
discussions on ribosomal sequence variation, Leo Beukeboom
and Kent Reed for reviewing the manuscript. This research was
supported by USDA 91-37302-6206 and NSF BSR8906231.
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