Field prevalence of Wolbachia in the mosquito vector Aedes albopictus

The endosymbiotic bacteria in the genus Wolbachia have been proposed as a potential candidate to deliver pathogen-blocking genes into natural populations of medically important insects. The successful application of Wolbachia in insect vector control depends on the ability of the agent to successfully invade and maintain itself at high frequency under field conditions. Here, we evaluated the prevalence of Wolbachia infections in a field population of the Wolbachia-superinfected mosquito Aedes albopictus. A field prevalence of 100% (n = 1,016) was found in a single population in eastern Thailand via polymerase chain reaction (PCR) testing of Wolbachia both from individual parent females and their corresponding F1 offspring. This is the first report of accurate Wolbachia prevalence in a field population of an insect disease vector. The prevalence of superinfection was estimated to be 99.41%. All single-infected individual mosquitoes (n = 6) were found to harbor group A Wolbachia. For this particular population, none was found to be single-infected with group B Wolbachia. Our results also show that PCR testing of field materials alone without checking F1 offspring overestimated the natural prevalence of single infection. Thus, the confirmation of infection status by means of F1 offspring was critical to the accurate estimates of Wolbachia prevalence under field conditions.

Determination of Wolbachia genome size by Pulsed-Field Gel Electrophoresis

Genome sizes of six different Wolbachia strains from insect and nematode hosts have been determined by pulsed-field gel electrophoresis of purified DNA both before and after digestion with rare-cutting restriction endonucleases. Enzymes SmaI, ApaI, AscI, and FseI cleaved the studied Wolbachia strains at a small number of sites and were used for the determination of the genome sizes of wMelPop, wMel, and wMelCS (each 1.36 Mb), wRi (1.66 Mb), wBma (1.1 Mb), and wDim (0.95 Mb). The Wolbachia genomes studied were all much smaller than the genomes of free-living bacteria such as Escherichia coli (4.7 Mb), as is typical for obligate intracellular bacteria. There was considerable genome size variability among Wolbachia strains, especially between the more parasitic A group Wolbachia infections of insects and the mutualistic C and D group infections of nematodes. The studies described here found no evidence for extrachromosomal plasmid DNA in any of the strains examined. They also indicated that the Wolbachia genome is circular.

Wolbachia neither induces nor suppresses transcripts encoding antimicrobial peptides

Wolbachia are intracellular maternally inherited microorganisms that are associated with reproductive abnormalities such as cytoplasmic incompatibility (CI), feminization and parthenogenesis in the various arthropod species they infect. Surveys indicate that these bacteria infect more than 16% of all insect species as well as isopods, mites and nematodes, making Wolbachia one of the most ubiquitous parasites yet described. However, nothing is known about the interactions of this bacterium with the host's immune system. We studied the expression of inducible antimicrobial markers in the adults of two Wolbachia infected insect species, Drosophila simulans and Aedes albopictus. The lack of available immune markers in the mosquito species led us to clone part of the defensin gene from this species, which was found to be very similar to the other mosquito defensins cloned from Anopheles gambiae and Aedes aegypti. Comparisons of the expression pattern of the antibacterial markers between Wolbachia-infected and cured lines, and also between bacteria-challenged and unchallenged adults indicated that Wolbachia does not either constitutively induce or suppress the transcription of these antibacterial genes. In addition, no difference in the transcription of these genes was found between double and single Wolbachia-infected strains or between strains in which Wolbachia has different tissue tropisms.

Maternal transmission efficiency of Wolbachia superinfections in Aedes albopictus populations in Thailand

We examined the transmission efficiency of 2 strains of Wolbachia bacteria that cause cytoplasmic incompatibility in field populations of Aedes albopictus by polymerase chain reaction assay. We found mainland and island populations throughout Thailand to be superinfected with group A and B bacteria. Of 320 Wolbachia-positive adult mosquitoes, 97.5% were infected with both groups. Single infected individuals of each Wolbachia group were encountered in nearly equal numbers. We screened 550 offspring from 80 field-collected mothers and found the transmission efficiency of group A Wolbachia to be 96.7% and that of group B Wolbachia to be 99.6%. Mothers that did not transmit both Wolbachia infections to all of their offspring were significantly larger in size than those with perfect transmission fidelity. We discuss our findings in relation to the prospects of the use of Wolbachia as a gene-driving mechanism.

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.

Evolution of Wolbachia pipientis transmission dynamics in insects

Wolbachia pipientis is an intracellular bacterial parasite of arthropods that enhances its transmission by manipulating host reproduction, most commonly by inducing cytoplasmic incompatibility. The discovery of isolates with modified cytoplasmic incompatibility phenotypes and others with novel virulence properties is an indication of the potential breadth of evolutionary strategies employed by 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.

Wolbachia infections and arthropod reproduction

Wolbachia is an intracellular bacterium that is almost exclusively maternally transmitted, and its reproductive effects favor transmission of the intracellular bacterial agent at the expense of the arthropod population that is not infected. Wolbachia can cause cytoplasmic incompatibility, parthenogenesis and feminization in many arthropods.

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.

Interspecific and intraspecific horizontal transfer of Wolbachia in Drosophila

Cytoplasmic incompatibility (CI) in Drosophila simulans is related to infection of the germ line by a rickettsial endosymbiont (genus Wolbachia). Wolbachia were transferred by microinjection of egg cytoplasm into uninfected eggs of both D. simulans and D. melanogaster to generate infected populations. Transinfected strains of D. melanogaster with lower densities of Wolbachia than the naturally infected D. simulans strain did not express high levels of CI. However, transinfected D. melanogaster egg cytoplasm, transferred back into D. simulans, generated infected populations that expressed CI at levels near those of the naturally infected strain. A transinfected D. melanogaster line selected for increased levels of CI expression also displayed increased symbiont densities. These data suggest that a threshold level of infection is required for normal expression of CI and that host factors help determine the density of the symbiont in the host.

hnRNP A2 selectively binds the cytoplasmic transport sequence of myelin basic protein mRNA

Segregation of mRNAs in the cytoplasm of polar cells has been demonstrated for proteins involved in Xenopus and Drosophila oogenesis, and for some proteins in somatic cells. It is assumed that vectorial transport of the messages is generally responsible for this localization. The mRNA encoding the basic protein of central nervous system myelin is selectively transported to the distal ends of the processes of oligodendrocytes, where it is anchored to the myelin membrane and translated. This transport is dependent on a 21-nucleotide cis-acting segment of the 3'-untranslated region (RTS). Proteins that bind to this cis-acting segment have now been isolated from extracts of rat brain. A group of six 35-42-kDa proteins bind to a 35-base oligoribonucleotide incorporating the RTS, but not to several oligoribonucleotides with the same composition but randomized sequences, thus establishing specificity for the base sequence in the RTS. The most abundant of these proteins has been identified, by Edman sequencing of tryptic peptides and mass spectroscopy, as heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a 36-kDa member of a family of proteins that are primarily, but not solely, intranuclear. This protein was most abundant in samples from rat brain and testis, with lower amounts in other tissues. It was separated from the other polypeptides by using reverse-phase HPLC and shown to retain preferential association with the RTS. In cultured oligodendrocytes, hnRNP A2 was demonstrated by confocal microscopy to be distributed throughout the nucleus, cell soma, and processes.

FRA10B structure reveals common elements in repeat expansion and chromosomal fragile site genesis

A common mechanism for chromosomal fragile site genesis is not yet apparent. Folate-sensitive fragile sites are expanded p(CCG)n repeats that arise from longer normal alleles. Distamycin A or bromodeoxyuridine-inducible fragile site FRA16B is an expanded AT-rich similar to 33 bp repeat; however, the relationship between normal and fragile site alleles is not known. Here, we report that bromodeoxyuridine-inducible, distamycin A-insensitive fragile site FRA10B is composed of expanded similar to 42 bp repeats. Differences in repeat motif length or composition between different FRA10B families indicate multiple independent expansion events. Some FRA10B alleles comprise a mixture of different expanded repeat motifs. FRA10B fragile site and long normal alleles share flanking polymorphisms. Somatic and intergenerational FRA10B repeat instability analogous to that found in expanded trinucleotide repeats supports dynamic mutation as a common mechanism for repeat expansion.

Comparative in vitro effects of closantel and selected beta-ketoamide anthelmintics on a gastrointestinal nematode and vertebrate liver cells

PNU-87407 and PrNU-88509, beta-ketoamide anthelmintics that are structurally related to each other and to the salicylanilide anthelmintic closantel, exhibit different anthelmintic spectra and apparent toxicity in mammals, The basis for this differential pharmacology was examined in experiments that measured motility and adenosine triphosphate (ATP) levels in larval and adult stages of the gastrointestinal nematode, Haemonchus contortus, and in a vertebrate liver cell line and mitochondria, PNU-87407 and PNU-88509 both exhibited functional cross-resistance with closantel in larval migration assays using closantel-resistant and -sensitive isolates of H, contortus. Each compound reduced motility and,ATP levels in cultured adult H. contortus in a concentration- and time-dependent manner: however, motility was reduced more rapidly by PNU-88509, and ATP levels were reduced by lower concentrations of closantel than the beta-ketoamides. Tension recordings from segments of adult H, contortus showed that PNU-88509 induces spastic paralysis, while PNU-87407 and closantel induce flaccid paralysis of the somatic musculature. Marked differences in the actions of these compounds were also observed in the mammalian preparations. In Chang liver cells, ATP levels were reduced after 3 h exposures to greater than or equal to 0.25 mu M PNU-87407 1 mu M closantel or 10 mu M PNU-88509, Reductions in ATP caused by PNU-88509 were completely reversible, while the effects of closantel and PNU-87407; were irreversible. PNU-87407, closantel and PNU-88509 uncoupled oxidative phosphorylation in isolated rat liver mitochondria, inhibiting the respiratory control index (with glutamate or succinate as substrate) by 50% at concentrations of 0.14, 0.9 and 7.6 mu M respectively.