LETTERS TO NATURE Molecular identification of Agrobacterium tumefaciens microorganisms associated Rickettsiarickettsh Rickettsia prowazeki with parthenogenesis Ehrlichia rislicii Ehrlichia sennetsu R. Stouthamer*t, J. A. J. Breeuwert, R. F. Luck* Ehrlichia canis & J. H. Werrent L Cowdria ruminatium Ehrlichia equi * Department of Entomology, Agricultural University, PO Box 8031, r Anaplasma marginale 6700 EH Wageningen, The Netherlands Department of Biology, University of Rochester, Rochester, Nasonia vitripennis New York 14627, USA Culex pipiens $ Department of Entomology, University of California, Riverside, Trichogramma deion California 92521, USA PM / CIM Drosophila simulans CYTOPLASMICALLY inherited microorganisms are widespread in Ephestia cautella insects and have been implicated as causes of female partheno- Musciditurax uniraplor genesis (females developing from unfertilized eggs) and cyto- 50 plasmic incompatibility' - ' 5. Normal sexual reproduction can be FIG. 1 Most parsimonious phylogenetic tree of parthenogenesis microorgan- restored by treatment with antibiotics". Sequence analysis of the i sms (PM) of Trichogramma deion ( Bautista strain) and Muscidifurax uni- 5.6 DNA encoding 16S ribosomal RNA has shown that cytoplasmic raptor, several cytoplasmic incompatibility microorgansisms ( CIM) and 21-23 incompatibility bacteria from diverse insect taxa are closely related other representatives of the alpha subdivision of the Proteobacteria . (they share >95% sequence similarity) and belong to the alpha The 16S rDNA sequences of PM (bold) and CIM are identified by the host subdivision of Proteobacteria5-7. Here we show that partheno- species from which they were isolated. Escherichia coli (gamma subdivision) genesis-associated bacteria from parasitoid Hymenoptera also fall was used as an outgroup. Sequences were manually aligned using regions 21 into this bacterial group, having up to 99% sequence similarity of the 16S gene that are conserved in eubacteria . The aligned sequences to some incompatibility microorganisms. Both incompatibility and of parthenogenesis microorganisms were 1,512 bases in length, including gaps. Gaps were treated as a 'fifth' base. The aligned sequence dataset parthenogenesis microorganisms alter host chromosome behaviour was analysed with PAUP 3.0 (ref. 20) using the branch and Bound algorithm during early mitotic divisions of the egg' }". Incompatibility bac- to find the shortest tree(s). Two parsimonious trees (length 1,195 bases) teria act by interfering with paternal chromosome incorporation were generated. They only differed in the positioning of the Drosophila in fertilized eggs, whereas parthenogenesis bacteria prevent simulans and Ephestia cautella microorganisms within the PM/CIM group. segregation of chromosomes in unfertilized eggs. These traits are This is a result of the fact that only a partial 16S sequence is available for adaptive for the microorganisms. On the basis of their sequence these two cytoplasmic incompatibility microorganisms. Sequences of 16S similarities, we conclude that parthenogenesis bacteria and cyto- ribosomal genes of the M. uniraptor and Trichogramma symbionts were plasmic incompatibility bacteria form a monophyletic group of determined using the following procedure. Total DNA was extracted from microorganisms that `specialize' in manipulating chromosome white pupae (100 of Trichogramma spp. and 5 of Muscidifurax) after these were surface-sterilized with 70% ethanol, thoroughly washed with sterile behaviour and reproduction of insects. water and homogenized in a Mini-Bead beater (Biospec). After precipitation Identification of insect bacterial symbionts has long been with ethanol, bacterial 16S rDNA was PCR-amplified using conserved 16S hampered by the inability to culture these fastidious prokaryotes. rDNA primers (fD2 and rP2; ref. 21). PCR product was then directly cloned But the development of the polymerase chain reaction (PCR) i nto T-tailed M13mp18 vector and sequenced by the Sanger method using and use of DNA sequence encoding 16S RNA (16S sequences) Sequenase vr. 2.0 kit (US Biochemical; for sequence primers, see refs 5, 27). i n microbial phylogeny' $ has made it possible to determine their The nucleotide sequence data of parthenogenesis bacteria will appear in phylogenetic position. We amplified and sequenced the bacterial Genbank under the following accession numbers; M. uniraptor, _02882; T 16S ribosomal DNA from six parthenogenetic strains of Tricho- cordubensis, _02883; T deion TX, 102884; T, pretiosum, _02885, T deion Mo, 102886; T. deion Ba, _02887; T Deion gramma wasps (three different species) and one parthenogenetic SD, _02888. strain of Muscidifurax uniraptor. Trichogramma wasps are i n the eggs of bisexual strains or those with genetically based minute (around 0.5 mm) parasites of insect eggs, primarily those parthenogenesis. All microbe-associated parthenogenetic strains of Lepidoptera. Muscidifurax uniraptor is a Pteromalid pupal examined have a cytogenetic mechanism of parthenogenesis parasitoid of houseflies. Each of these strains harbours micro- (gamete duplication) that differs from those with genetic organisms associated with parthenogenesis. To check for PCR parthenogenesis (ref. 19; R. S. and D. J. Kazmer, unpublished amplification of bacteria not associated with parthenogenesis, data). sexual strains of Trichogramma were used as a control. Some Phylogenetic analysis of the 16S rDNA sequences from the forms of parthenogenesis in Trichogramma are genetically parthenogenesis microorganisms was undertaken using the par- based and are not associated with microorganisms'''. As a simony algorithm Branch and Bound of PAUP 3.0 (ref. 20). All second control, three strains of Trichogramma with parthenogenesis bacteria fall in the alpha subdivision of Proteo- genetic parthenogenesis were also examined for cytoplasmic bacteria, and form a group with over 95% sequence similarity microorganisms. For M. uniraptor, a closely related sexual ( Figs 1 and 2). Most interestingly, all parthenogenesis micro- species (M. raptor) was used as a PCR control. In addition, organisms are closely related to cytoplasmic incompatibility standard controls for PCR contamination were set up. PCR bacteria, which are found in diverse insect taxa including beetles products were obtained from all microbe-associated ( Coleoptera), butterflies (Lepidoptera), flies (Diptera) and 5- parthenogenetic T. deion (four different North American collec- wasps (Hymenoptera) '. Wolbachia pipientis, the cytoplasmic tions originating from South Dakota, Texas, and two different i ncompatibility microbe of Culex pipiens, is type species of the l ocalities in California: Bautista Canyon and Mountain Center), genus Wolbachia (see ref. 6). Based on their high sequence T. pretiosum (from Mexico), T. cordubensis (from Spain) and si milarity, it is reasonable also to place parthenogenesis bacteria M. uniraptor. No DNA was amplified from the controls. within this genus. The results from PCR are consistent with those from Figure 1 shows the phylogenetic position of the partheno- cytogenetic and antibiotic studies. Bacteria are easily visualized genesis/incompatibility group relative to several representatives i n the eggs of parthenogenetic Trichogramma strains4 , and anti- of the alpha subdivision of proteobacteria. The parthenogenesis/ biotic treatment results in reversion to sexual reproduction and incompatibility ( Wolbachia) bacteria form a monophyletic elimination of the microorganisms' '4 . Bacteria are not present group relative to other alpha Proteobacteria. The closest sequen- 66 NATURE - VOL 361 - 7 JANUARY 1993 LETTERS TO NATURE ced relatives to this group are Anaplasma marginaleZt , Cowdria TABLE 1 Diagnostic nucleotide of subgroups of the ruminantium 22 , and several species of Ehrlichia 23 . Each of these positions parthenogenesis and incompat bility bacteria are arthropod vectored microorganisms that cause mammalian Type 1 CIM Type 2 diseases. M CIM/PM A more detailed phylogenetic analysis of the parthenogenesis/ (4 species) (3 specie PPosition , 6 strains) (4 species) incompatibility group is shown in Fig. 2. Although partial 16S sequences (the first 700 bases) are known for incompatibility 649 r A G A bacteria from several insect species6 7 , nearly complete sequen- 650 L - T __ C T A A G ces have only been published for incompatibility bacteria of the 693 6 s 760 A A G mosquito Culex pipienS and wasps of the genus Nasonia . This 844 - T is unfortunate, because much of the phylogenetically informa- 1,037 A A G tive sequence information is in the second half (3' end) of the 1,047 A A G 16S rDNA gene s . Therefore we did the phylogenetic analysis 1,127 G_ A for those incompatibility microbe sequences that are nearly 1,129 1 C _C J complete, including Culex' and Nasonia incompatibility bac- 1,143 A A G l 1,144A T T teria . 1,210 T T C The parthenogenesis/ incompatibility group is divided into 1,245 T T G two subgroups that differ consistently from each other at 15 1,262 G G A positions over 1,464 bases of the almost complete 16S ribosomal 1,268 A A G gene. The two subgroups represent real 16S variants based on 1,285 T analysis of secondary structures . The diagnostic base positions 1,292 A A C separating the two subgroups are shown in Table 1. Although 1,327 A A G incompatibility microorganisms from different insects occur in 1,364 T T C both subgroups, all parthenogenesis microorganisms from 1,442 G G A 1,457 A A G Trichogramma wasps fall into subgroup I. All 16S rDNA