Wolbachia: why these bacteria are important to genome research

Intracellular bacteria of the genus Wolbachia were first discovered in mosquitoes in the 1920s. Their superficial similarity to pathogenic rickettsia initially raised interest in them as potential human pathogens. However, injection experiments with mice showed that they were non-pathogenic, and they were subsequently classified as symbionts of insects. Until the 1970s, Wolbachia was considered to infect a limited number of species of mosquitoes. It is now clear that Wolbachia is an extremely common intracellular agent of invertebrates, infecting nearly all the major groups of arthropods and other terrestrial invertebrates. Its wide host range and abundance can be attributed partly to the unusual phenotypes it exerts on the host it infects. These include the induction of parthenogenesis (the production of female offspring from unmated mothers) in certain insects, the feminization of genetic male crustaceans to functional phenotypic females, and the failure of fertilization in hosts when males and females have a different infection status (cytoplasmic incompatibility). All of these phenotypes favor maternal transmission of the intracellular Wolbachia. In the last year, Wolbachia has also been shown to be a widespread symbiont of filarial nematodes. It appears that Wolbachia is needed by the adult worm for normal fertility, indicating that Wolbachia is behaving like a classic mutualist in this case. This discovery exemplifies that the extent of the host range of Wolbachia and its associated phenotypes is still far from fully understood.

Wolbachia genomes and the many faces of symbiosis

How could scientists working on the pathogenesis of filarial diseases, speciation and parthenogenesis in insects, sex-ratio deviations in crustaceans, pest control, and the evolution of bacterial genomes be united? How could a common research project attract the interest of these scientists? How could parasitology be made even more multi-disciplinary? Two workshops organized by New England Biolabs Inc. (Beverly, MA, USA) provide a simple answer to these questions: studying the genomes of Wolbachia endosymbionts.

Wolbachia infections are distributed throughout insect somatic and germ line tissues

Wolbachia are intracellular microorganisms that form maternally-inherited infections within numerous arthropod species. These bacteria have drawn much attention, due in part to the reproductive alterations that they induce in their hosts including cytoplasmic incompatibility (CI), feminization and parthenogenesis. Although Wolbachia's presence within insect reproductive tissues has been well described, relatively few studies have examined the extent to which Wolbachia infects other tissues. We have examined Wolbachia tissue tropism in a number of representative insect hosts by western blot, dot blot hybridization and diagnostic PCR. Results from these studies indicate that Wolbachia are much more widely distributed in host tissues than previously appreciated. Furthermore, the distribution of Wolbachia in somatic tissues varied between different Wolbachia/host associations. Some associations showed Wolbachia disseminated throughout most tissues while others appeared to be much more restricted, being predominantly limited to the reproductive tissues. We discuss the relevance of these infection patterns to the evolution of Wolbachia/host symbioses and to potential applied uses of Wolbachia.

Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis

The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.

Rescuing Wolbachia have been overlooked ...

Comment: Wolbachia, intracellular bacteria transmitted through the egg, have been estimated to infect more than 16% of all insect species, as well as other arthropods. They distort their hosts' reproduction, inducing parthenogenesis, feminization and cytoplasmic incompatibility. This favours the reproduction of infected female hosts at the expense of uninfected females. Here we show that several Wolbachia strains that cannot generate modifications in host sperm can still rescue the modifications caused by other strains as long as the two strains are sufficiently closely related.

In vitro cultivation of Wolbachia pipientis in an Aedes albopictus cell line

A continuous cell line, Aa23, was established from eggs of a strain of the Asian tiger mosquito, Aedes albopictus, naturally infected with the intracellular symbiont Wolbachia pipientis. The resulting cell line was shown to be persistently infected with the bacterial endosymbiont. Treatment with antibiotics cured the cells of the infection. In the course of establishing this cell line it was noticed that RFLPs in the PCR products of two Wolbachia genes from the parental mosquitoes were fixed in the infected cell line. This indicates that the mosquito host was naturally superinfected with different Wolbachia strains, whereas the infected cell line derived from these mosquitoes only contained one of the original Wolbachia strains. The development of anin vitroculture system for this fastidious microorganism should facilitate molecular analysis of the reproduction distorting phenotypes it induces in natural arthropod hosts.

Replacement of the natural Wolbachia symbiont of Drosophila simulans with a mosquito counterpart

Inherited rickettsial symbionts of the genus Wolbachia occur commonly in arthropods and have been implicated in the expression of parthenogenesis, feminization and cytoplasmic incompatibility phenomena in their respective hosts. Here we use purified Wolbachia from the Asian tiger mosquito, Aedes albopictus, to replace the natural infection of Drosophila simulans by means of embryonic microinjection techniques. The transferred Wolbachia infection behaves like a natural Drosophila infection with regard to its inheritance, cytoskeleton interactions and ability to induce incompatibility when crossed with uninfected flies. The transinfected flies are bidirectionally incompatible with all other naturally infected strains of Drosophila simulans, however, and as such represent a unique crossing type. The successful transfer of this symbiont between distantly related hosts suggests that it may be possible to introduce this agent experimentally into arthropod species of medical and agricultural importance in order to manipulate natural populations genetically.

16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects

Bacterial endosymbionts of insects have long been implicated in the phenomenon of cytoplasmic incompatibility, in which certain crosses between symbiont-infected individuals lead to embryonic death or sex ratio distortion. The taxonomic position of these bacteria has, however, not been known with any certainty. Similarly, the relatedness of the bacteria infecting various insect hosts has been unclear. The inability to grow these bacteria on defined cell-free medium has been the major factor underlying these uncertainties. We circumvented this problem by selective PCR amplification and subsequent sequencing of the symbiont 16S rRNA genes directly from infected insect tissue. Maximum parsimony analysis of these sequences indicates that the symbionts belong in the α-subdivision of the Proteobacteria, where they are most closely related to the Rickettsia and their relatives. They are all closely related to each other and are assigned to the type species Wolbachia pipientis. Lack of congruence between the phylogeny of the symbionts and their insect hosts suggests that horizontal transfer of symbionts between insect species may occur. Comparison of the sequences for W. pipientis and for Wolbachia persica, an endosymbiont of ticks, shows that the genus Wolbachia is polyphyletic. A PCR assay based on 16S primers was designed for the detection of W. pipientis in insect tissue, and initial screening of insects indicates that cytoplasmic incompatibility may be a more general phenomenon in insects than is currently recognized.

Erluanbi, Southern Taiwan

Erluanbi is the most southern tip of Taiwan (Formosa) where the Taiwan (Formosa) Strait meets the Pacific Ocean. The Erluanbi national park is renown for its lighthouse, and its coral reef, and it hosts also some prehistoric sites bating back to 5,000 to 6,500 years. The Erluanbi (or Eluan Pi) lighthouse was completed in 1883, following requests from the American and Japanese governments to the Chinese government after several ship wrecks in the 1860s. Chinese troops were sent to protect the lighthouse construction from attacks by local tribesmen, and the lighthouse was surrounded a small fort with cannons and a ditch to protect it. It is a rare example of a fortified lighthouse in the world. The lighthouse itself is 21.4 m high and its light is 56.4 m above high water. The light flashes every 10 seconds and its range is 27.2 nautical miles. The surrounding Erluanbi national park is located on a raised coral reef with some huge fringing reef : e.g., the "sea pavillon". With the topical oceanic climate, the elevated reef hosts an unique vegetation and ecology. Since 1956, numerous prehistoric artefacts were uncovered including stone slab coffins and pottery (plain and painted), that encompassed at least four cultural stages from BC 4,500 to AD 800.

Glossocercus chelodinae (MacCallum, 1921) n. comb. (Cestoda : Dilepididae) from freshwater turtles in Australia and a redefinition of the genus Bancroftiella Johnston, 1911

Glossocercus chelodinae (MacCallum, 1921) n. comb. is redescribed from fresh material recovered from the intestine of an Australian freshwater turtle, Chelodina expansa. G. chelodinae can be distinguished from all other species of the genus by the shape of its rostellar hooks. it is suggested that this species has colonised fish-eating turtles from fish-eating birds. The morphological relationships among Parvitaenia, Bancroftiella and Glossocercus are discussed. The diagnosis of Bancroftiella is amended and marsupials are eliminated as hosts. Bancroftiella sudarikovi Spasskii & Yurpalova, 1970 becomes a synonym of Glossocercus glandularis (Fuhrmann, 1905); only B. tennis Johnston, 1911, the type-species, and B. ardeae Johnston, 1911 remain in the genus.

The diversity of the Digenea of Australian animals

The Digenea is one of five major helminth assemblages represented in Australian animals. History of the study of digeneans in Australia is reviewed briefly to show that it has never been subjected to the kind of sustained study needed to reach an understanding of it. The Australian vertebrate fauna comprises over 5500 species. These have so far been shown to harbour just over 70 families, about 306 genera and 566 species of digeneans. Digeneans occur in all classes of vertebrates in Australia but are distributed very unevenly; aquatic hosts are generally most heavily infected, but many terrestrial species are also infected. Particular weaknesses in knowledge of the fauna concern the bats, cetaceans and teleosts. Another weakness is in knowledge of life-cycles; representative life-cycles are known for only about 20 of the 70 families known in Australia. Estimates of the overall size of the fauna are dependent on an understanding of sampling strategies, the heterogeneity of distribution of the fauna, and the nature of host-specificity. These subjects are reviewed briefly and an estimate of the total fauna is made. There may be as many as 6000 species of digeneans in Australia. (C) 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd.

From sabotage to camouflage: viral evasion of cytotoxic T lymphocyte and natural killer cell-mediated immunity

The outcome of a virus infection is strongly influenced by interactions between host immune defences and virus 'anti-defence' mechanisms. For many viruses, their continued survival depends on, the speed of their attach: their capacity to replicate and transmit to uninfected hosts prior to their elimination by an effective immune response. In contrast, the success of persistent viruses lies in their capacity for immunological subterfuge: the evasion of host defence mechanisms by either mutation (covered elsewhere in this issue, by Gould and Bangham, pp. 321-328) or interference with the action of host cellular proteins that are important components of the immune response. This review will focus on the strategies employed by persistent viruses against two formidable host defences against virus infection: the CD8+ cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses.

Type 1 and Type 2 cytokines: from basic science to fungal infections

At the present time, it is clear that Th1 responses afford protection against the fungi; however, the development, maintenance and function of the protective immune responses are complex mechanisms and are influenced by multiple factors. The route of infection has been shown to affect initial cytokine production and, consequently, the induction of protective Th1 responses. The ability of different isolates of the same fungal agent to induce and sustain a protective response has also been emphasized. Protective immune responses have been shown to vary in genetically different mouse strains after infection. In addition, these protective responses, such as cellular influx and cytokine production, also vary within the same animal depending on the tissue infected. The functional dominance of certain cytokines over others in influencing development and maintenance of protective responses has been discussed. Certain cytokines may act differently in hosts lacking important components of their innate or immune repertoire. It is evident from these presentations that a more comprehensive understanding of the protective mechanisms against different fungal agents is emerging. However, there is still much to learn before cytokine modulatory therapy can be used effectively without risk in the human host.

First record of Phytophthora capsici from Queensland

Phytophthora capsici has been identified from two new hosts in Queensland, Custard Apple (Annona squamosa) and Mandevilla sp. This is the first record of P. capsici in Queensland and only the second record of the pathogen. in Australia. It is also the first report of the pathogen on these hosts.

V4 region of small subunit rDNA indicates polyphyly of the Fellodistomidae (Digenea) which is supported by morphology and life-cycle data

There is no morphological synapomorphy for the disparate digeneans, the Fellodistomidae Nicoll, 1909. Although all known life-cycles of the group include bivalves as first intermediate hosts, there is no convincing morphological synapomorphy that can be used to unite the group. Sequences from the V4 region of small subunit (18S) rRNA genes were used to infer phylogenetic relationships among 13 species of Fellodistomidae from four subfamilies and eight species from seven other digenean families: Bivesiculidae; Brachylaimidae; Bucephalidae; Gorgoderidae; Gymnophallidae; Opecoelidae; and Zoogonidae. Outgroup comparison was made initially with an aspidogastrean. Various species from the other digenean families were used as outgroups in subsequent analyses. Three methods of analysis indicated polyphyly of the Fellodistomidae and at least two independent radiations of the subfamilies, such that they were more closely associated with other digeneans than to each other. The Tandanicolinae was monophyletic (100% bootstrap support) and was weakly associated with the Gymnophallidae (< 50-55% bootstrap support). Monophyly of the Baccigerinae was supported with 78-87% bootstrap support, and monophyly of the Zoogonidae + Baccigerinae received 77-86% support. The remaining fellodistomid species, Fellodistomum fellis, F. agnotum and Coomera brayi (Fellodistominae) plus Proctoeces maculatus and Complexobursa sp. (Proctoecinae), formed a separate clade with 74-92% bootstrap support. On the basis of molecular, morphological and life-cycle evidence, the subfamilies Baccigerinae and Tandanicolinae are removed from the Fellodistomidae and promoted to familial status. The Baccigerinae is promoted under the senior synonym Faustulidae Poche, 1926, and the Echinobrevicecinae Dronen, Blend & McEachran, 1994 is synonymised with the Faustulidae. Consequently, species that were formerly in the Fellodistomidae are now distributed in three families: Fellodistomidae; Faustulidae (syn. Baccigerinae Yamaguti, 1954); and Tandanicolidae Johnston, 1927. We infer that the use of bivalves as intermediate hosts by this broad range of families indicates multiple host-switching events within the radiation of the Digenea.