The Genus Wolbachia

Numerous invertebrate species form long lasting symbioses with bacteria (Buchner, 1949; Buchner, 1965). One of the most common of these bacterial symbionts is Wolbachia pipientis, which has been estimated to infect anywhere from 15–75% of all insect species (Werren et al., 1995a; West et al., 1998; Jeyaprakash and Hoy, 2000; Werren and Windsor, 2000) as well as many species of arachnids, terrestrial crustaceans and filarial nematodes (O’Neill et al., 1997a; Bandi et al., 1998). In most arthropod associations, Wolbachia act as reproductive parasites manipulating the reproduction of their hosts to enhance their own vertical transmission. There appears to be little direct fitness cost to the infected host besides the costs arising from the reproductive manipulations. However instances have been reported where Wolbachia can be either deleterious (Min and Benzer, 1997; Bouchon et al., 1998) or beneficial (Girin and Boultreau, 1995; Stolk and Stouthamer, 1995; Wade and Chang, 1995; Vavre et al., 1999b; Dedeine et al., 2001) to their hosts. Wolbachia were first described as intracellular Rickettsia-like organisms (RLOs), infecting the gonad cells of the mosquito, Culex pipiens (Hertig and Wolbach, 1924), and were later named 'Wolbachia pipientis' (Hertig, 1936). It was not until the work of Yen and Barr (Yen and Barr, 1971; Yen and Barr, 1973) that Wolbachia were implicated in causing crossing incompatibilities between different mosquito populations (Laven, 1951; Ghelelovitch, 1952). When polymerase chain reaction (PCR) diagnostics for Wolbachia became available, it became clear that this agent was both extremely widespread and also responsible for a range of different reproductive phenotypes in the different hosts it infected (O’Neill et al., 1992; Rousset et al., 1992; Stouthamer et al., 1993). The most common of these are cytoplasmic incompatibility, inducing parthenogenesis, overriding host sex-determination, and male-killing (O’Neill et al., 1997a). As of the time of this writing, more than 450 different Wolbachia strains with unique gene sequences, different phenotypes, and infecting different hosts have been deposited in GenBank and the Wolbachia host database (http://www.wolbachia.sols. uq.edu.au).

Molecular phylogeny of Wolbachia endosymbionts in southeast asian mosquitoes (Diptera: Culicidae) based on wsp gene sequences

Wolbachia are maternally inherited intracellular bacteria that infect a wide range of arthropods and nematodes and are associated with various reproductive abnormalities in their hosts. Insect-associated Wolbachia form a monophyletic clade in the α-Proteobacteria and recently have been separated into two supergroups (A and B) and 19 groups. Our recent polymerase chain reaction (PCR) survey using wsp specific primers indicated that various strains of Wolbachia were present in mosquitoes collected from Southeast Asia. Here, we report the phylogenetic relationship of the Wolbachia strains found in these mosquitoes using wsp gene sequences. Our phylogenetic analysis revealed eight new Wolbachia strains, five in the A supergroup and three in the B supergroup. Most of the Wolbachia strains present in Southeast Asian mosquitoes belong to the established Mors, Con, and Pip groups.

Wolbachia infections of tephritid fruit flies: molecular evidence for five distinct strains in a single host species

Endosymbiotic bacteria of the genus Wolbachia are widespread among arthropods and can induce cytoplasmic incompatibility, thelytokous parthenogenesis, male-killing or feminization in their hosts. Here, we report phylogenetic relationships of Wolbachia in tephritid fruit flies based on wsp gene sequences. We also report, for the first time, five distinct strains of Wolbachia in Bactrocera ascita sp. B. Four of the five Wolbachia strains found in this species were in the same groups as those found in other tephritid fruit flies, suggesting possible horizontal transmission of Wolbachia from other fruit flies into B. ascita sp. B. The unreliability of wsp-specific group primers demonstrated in this study suggests that these primers might be useful only for preliminary identification of Wolbachia. Final determination of group affiliation needs to be verified with wsp sequence data.

wsp Gene sequences from the Wolbachia of filarial nematodes

Wolbachia endosymbiotic bacteria are widespread in arthropods and are also present in filarial nematodes. Almost all filarial species so far examined have been found to harbor these endosymbionts. The sequences of only three genes have been published for nematode Wolbachia (i.e., the genes coding for the proteins FtsZ and catalase and for 16S rRNA). Here we present the sequences of the genes coding for the Wolbachia surface protein (WSP) from the endosymbionts of eight species of filaria. Complete gene sequences were obtained from the endosymbionts of two different species, Dirofilaria immitis and Brugia malayi. These sequences allowed us to design general primers for amplification of the wsp gene from the Wolbachia of all filarial species examined. For these species, partial WSP sequences (about 600 base pairs) were obtained with these primers. Phylogenetic analysis groups these nematode wsp sequences into a coherent cluster. Within the nematode cluster, wsp-based Wolbachia phylogeny matches a previous phylogeny obtained with ftsZ gene sequences, with a good consistency of the phylogeny of hosts (nematodes) and symbionts (Wolbachia). In addition, different individuals of the same host species (Dirofilaria immitis and Wuchereria bancrofti) show identical wsp gene sequences.

The novel genome organization of the insect picorna-like virus Drosophila C virus suggests this virus belongs to a previously undescribed virus family

The complete nucleotide sequence of the genomic RNA from the insect picorna-like virus Drosophila C virus (DCV) was determined. The DCV sequence predicts a genome organization different to that of other RNA virus families whose sequences are known. The single-stranded positive-sense genomic RNA is 9264 nucleotides in length and contains two large open reading frames (ORFs) which are separated by 191 nucleotides. The 5' ORF contains regions of similarities with the RNA-dependent RNA polymerase, helicase and protease domains of viruses from the picornavirus, comovirus and sequivirus families. The 3' ORF encodes the capsid proteins as confirmed by N-terminal sequence analysis of these proteins. The capsid protein coding region is unusual in two ways: firstly the cistron appears to lack an initiating methionine and secondly no subgenomic RNA is produced, suggesting that the proteins may be translated through internal initiation of translation from the genomic length RNA. The finding of this novel genome organization for DCV shows that this virus is not a member of the Picornaviridae as previously thought, but belongs to a distinct and hitherto unrecognized virus family.

Carrageenans from Australian representatives of the family Cystocloniaceae (Gigartinales, Rhodophyta), with description of Calliblepharis celatospora sp. nov., and transfer of Austroclonium to the family Areschougiaceae

Ten Australian representatives from seven of the 10 genera presently constituting the family Cystolcloniaceae have been analyzed for their cell-wall galactans. Included in our survey are the monotypic Australian-endemic genera Austroclonium, Gloiophyllis, Erythronaema, and Stictosporum, one species of Craspedocarpus, three species of Rhodophyllis, and two species of Calliblepharis. As one of the species of the latter genus is endemic to Western Australia and presently undescribed, we illustrate its habit and anatomical features in formally proposing to name it Calliblepharis celatospora Kraft, sp. nov. All the species surveyed essentially produce typical iota (iota)-carrageenans, with the exception of Austroclonium. The sulfated galactans from Austroclonium predominantly contain the repeating units of iota-, alpha (alpha)-, and 6'-O-methylated iota- and alpha-carrageenans; whether these exist as discrete polysaccharides or a complex hybrid structure was not resolved. Thus, Austroclonium carrageenans resemble the polysaccharides from Rhabdonia, Areschougia, and Erythroclonium. Although these latter three genera are currently included in the large gigartinalean family Solieriaceae, all produce significantly different carrageenans from Solieria itself and related genera such as Eucheuma, Kappaphycus, Betaphycus, Sarcodiotheca, Agardhiella, Sarconema, and Callophycus. In consideration of these findings, as well as of significant anatomical similarities, we provisionally recommend reestablishment of the family Rhabdoniaceae Kylin (as the family Areschougiaceae J. Agardh) for Rhabdonia, Areschougia, Erythroclonium, and Austroclonium.

Morphological and biological notes on Polymorphus (Profilicollis) sphaerocephalus and Corynosoma stanleyi (Polymorphidae : Acanthocephala)

Polymorphus (Profilicollis) sphaerocephalus (Bremser in Rudolphi, 1819) Van Cleave, 1947 (Polymorphidae) cystacanths were recovered from 5 species of grapsid crabs (Paragrapsus gaimardii (Milne Edwards, 1837), Paragrapsus laevis (Dana, 1852), Paragrapsus quadridentatus (Milne Edwards, 1837), Brachynotus spinosus (Milne Edwards, 1853), and Cyclograpsus granulosus (Milne Edwards, 1853)) and 1 species of portunid crab (Nectocarcinus integrifrons (Linnaeus, 1766)) from intertidal zones in southern temperate waters of Australia. Cystacanths of Corynosoma stanleyi Smales, 1986 (Polymorphidae) were also recovered from P. gaimardii, P. quadridentatus, and C. granulosus. Polymorphus (P.) sphaerocephalus was the most prevalent (100%) in C. granulosus at Flinders I. and C. stanleyi was most prevalent (59.1%) in C. granulosus at Dunally Channel, Tasmania.

Phylogenetic position of Chitinophaga pinensis in the Flexibacter-Bacteroides-Cytophaga phylum

Comparison of the 16S rRNA gene sequence determined for Chitinophaga pinensis showed that this species is most closely related to Flexibacter filiformis in the Flexibacter-Bacteroides-Cytophaga phylum, These two chitinolytic bacteria, which are characterized by transformation into spherical bodies on ageing, belong to a strongly supported lineage that also includes Cytophaga arvensicola, Flavobacterium ferrugineum and Flexibacter sancti, The lineage is distinct from the microcyst-forming species Sporocytophaga myxococcoides.

The phylogenetic relationships of Caulobacter, Asticcacaulis and Brevundimonas species and their taxonomic implications

The phylogenetic relationships among the species of Caulobacter, Asticcacaulis and Brevundimonas were studied by comparison of their 16S rDNA sequences. The analysis of almost complete sequences confirmed the early evolutionary divergence of the freshwater and marine species of Caulobacter reported previously [Stahl, D. A., Key, R,, Flesher, B, & Smit, J. (1992), J Bacteriol 174, 2193-2198]. The freshwater species formed two distinct clusters. One cluster contained the species Caulobacter bacteroides, Caulobacter crescentus, Caulobacter fusiformis and Caulobacter henricii. C, bacteroides and C, fusiformis are very closely related (sequence identity 99.8%). The second cluster was not exclusive and contained the species Caulobacter intermedius, Caulobacter subvibrioides and Caulobacter variabilis, as well as Brevundimonas diminuta and Brevundimonas vesicularis, The marine species Caulobacter halobacteroides and Caulobacter maris were very closely related, with a sequence identity of 99.7%, These two species were most closely but distantly related to the marine hyphal/budding bacteria Hyphomonas jannaschiana and Hirschia baltica, which formed a deep phylogenetic line with Rhodobacter sphaeroides and Rhodobacter capsulatus, Caulobacter leidyia is unrelated to the other species of Caulobacter and belongs to the alpha-4 subclass of the Proteobacteria, forming a distinct cluster with Asticcacaulis excentricus and Asticcacaulis biprosthecium, The taxonomic implications of the polyphyletic nature of the genus Caulobacter and the absence of a type culture for the type species of the genus, Caulobacter vibrioides, are discussed.

Description of Skermanella parooensis gen. nov., sp. nov. to accommodate Conglomeromonas largomobilis subsp. parooensis following the transfer of Conglomeromonas largomobilis subsp. largomobilis to the genus Azospirillum

As a consequence of the transfer of the type species Conglomeromonas largomobilis subsp. largomobilis to the genus Azospirillum, the name of the genus Conglomeromonas must be changed in accordance with Rule 37a(1) of the International Code of Nomenclature of Bacteria. Consequently, it is proposed that the subspecies Conglomeromonas largomobilis subsp, parooensis be transferred to the genus Skermanella gen, nov. as the type species Skermanella parooensis gen, nov., sp, nov. This taxon belongs to an isolated subline of descent in the Azospirillum branch of the alpha-Proteobacteria. The spelling of the specific epithet of Azospirillum largomobile is corrected to Azospirillum largimobile.

Phylogenetic relationships among members of the Comamonadaceae, and description of Delftia acidovorans (den Dooren de Jong 1926 and Tamaoka et al. 1987) gen. nov., comb. nov.

The phylogenetic relationships among members of the family Comamonadaceae and several unclassified strains were studied by direct sequencing of their PCR-amplified 16S rRNA genes. Based on the 16S rRNA gene sequence analysis, members of the family formed a coherent group. The closest relatives are species of the Rubrivivax sub-group: Leptothrix discophora, Ideonella dechloratans and Rubrivivax gelatinosus. The genus Hydrogenophaga formed two subclusters, as did the species of Acidovorax, whereas the five species of the genus [Aquaspirillum] were polyphyletic. Comamonas acidovorans was phylogenetically distant from the type species of Comamonas, Comamonas terrigena. On the basis of this work and previous studies, Comamonas acidovorans is removed from the genus Comamonas and renamed as Delftia acidovorans gen. nov., comb, nov. Descriptions of the new genus Delftia and of the type species Delftia acidovorans, for which the type strain is ATCC 15668(T), are presented.

Molecular characterization of Drosophila C virus isolates

Reverse transcription coupled with polymerase chain reaction and restriction enzyme analysis was used to characterize 12 Drosophila C virus isolates from geographically different regions. A 1.2-kb fragment was amplified from cDNA and profiles from digestion with 20 restriction enzymes were generated. Analysis of the restriction fragment data gave estimates of nucleotide divergence of 0-10% between isolates. The isolates were grouped on the basis of genetic distance estimates derived from the restriction data. For the isolates from which a single genotype could be purified, a geographical pattern in the distribution of viral genotypes was identified. The 4 Moroccan isolates were very closely related to each other, differing in only 1 restriction profile. The 2 Australian isolates were each other's closest relatives, as were the 2 isolates first recovered in France. The PCR-RFLP technique used in this study has provided us with a simple procedure which can be used to characterize DCV isolates. A single enzyme, Tag I, generated 5 distinct and diagnostic restriction fragment patterns, which allowed easy assignment of isolates to one of the five viral genotypes identified in this study. (C) 1999 Academic Press.