Supergroup C Wolbachia,mutualist symbionts of filarial nematodes,have a distinct genome structure

Wolbachia pipientis is possibly the most widespread endosymbiont of arthropods and nematodes. While all Wolbachia strains have historically been defined as a single species, 16 monophyletic clusters of diversity (called supergroups) have been described. Different supergroups have distinct host ranges and symbiotic relationships, ranging from mutualism to reproductive manipulation. In filarial nematodes, which include parasites responsible for major diseases of humans (such as Onchocerca volvulus, agent of river blindness) and companion animals (Dirofilaria immitis, the dog heartworm), Wolbachia has an obligate mutualist role and is the target of new treatment regimens. Here, we compare the genomes of eight Wolbachia strains, spanning the diversity of the major supergroups (A–F), analysing synteny, transposable element content, GC skew and gene loss or gain. We detected genomic features that differ between Wolbachia supergroups, most notably in the C and D clades from filarial nematodes. In particular, strains from supergroup C (symbionts of O. volvulus and D. immitis) present a pattern of GC skew, conserved synteny and lack of transposable elements, unique in the Wolbachia genus. These features could be the consequence of a distinct symbiotic relationship between C Wolbachia strains and their hosts, highlighting underappreciated differences between the mutualistic supergroups found within filarial nematodes.     

Spontaneous inflammatory pain model from a mouse line with N-ethyl-N-nitrosourea mutagenesis

Background

Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi.

Methods

Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles.

Results and discussion

Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms) and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%). In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes). Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton) were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion).

Conclusions

Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron-transport, metabolism, anti-oxidants, and others with unknown functions had increased expression signals after doxycycline treatment. These results suggest that female worms are able to compensate in part for the loss of Wolbachia so that they can survive, albeit without reproductive capacity. This study of doxycycline induced changes in gene expression has provided new clues regarding the symbiotic relationship between Wolbachia and B. malayi.     

Detection and characterization of Wolbachia infection in silkworm

Wolbachia naturally infects a wide variety of arthropods, where it plays important roles in host reproduction. It was previously reported that Wolbachia did not infect silkworm. By means of PCR and sequencing we found in this study that Wolbachia is indeed present in silkworm. Phylogenetic analysis indicates that Wolbachia infection in silkworm may have occurred via transfer from parasitic wasps. Furthermore, Southern blotting results suggest a lateral transfer of the wsp gene into the genomes of some wild silkworms. By antibiotic treatments, we found that tetracycline and ciprofloxacin can eliminate Wolbachia in the silkworm and Wolbachia is important to ovary development of silkworm. These results provide clues towards a more comprehensive understanding of the interaction between Wolbachia and silkworm and possibly other lepidopteran insects.     

Phylogenetically distinct Wolbachia gene and pseudogene sequences obtained from the African onchocerciasis vector Simulium squamosum

Wolbachia are intracellular bacteria mostly found in a diverse range of arthropods and filarial nematodes. They have been classified into seven distinct ‘supergroups’ and other lineages on the basis of molecular phylogenetics. The arthropod-infecting Wolbachia are usually regarded as reproductive parasites because they manipulate their host species’ sexing system to enhance their own spread, and this has led to their investigation as potential agents of genetic control in medical entomology. We report 12 partial Wolbachia gene sequences from: aspC, aspS, dnaA, fbpA, ftsZ, GroEL, hcpA, IDA, rpoB, rpe, TopI and wsp as well as a single ftsZ pseudogene sequence, which have all been PCR-amplified from Simulium squamosum (Diptera: Simuliidae). To our knowledge this is the first such report from Simuliidae. Uninterrupted open-reading frame sequences were obtained from all 12 genes, covering ∼6.2 kb of unique DNA sequence. Phylogenetic analyses with the different coding genes gave consistent results suggesting that the Wolbachia sequences obtained here do not derive from any of the known Wolbachia supergroups or lineages. Consistent with a unique genetic status for the S. squamosumWolbachia, the hypervariable regions of the Wolbachia-specific wsp gene were distinct from all previous records in both sequence and length. As well as potential implications for newly emerging Wolbachia-based disease control methods, the results may be relevant to some problems experienced in the laboratory colonisation of Simulium damnosum sensu lato and why it is such a diverse species complex.     

Distribution and Evolutionary Impact of Wolbachia on Butterfly Hosts

Wolbachia are maternally inherited endosymbiotic alpha-proteobacteria found in terrestrial arthropods and filarial nematodes. They are transmitted vertically through host cytoplasm and alter host biology by inducing various reproductive alterations, like feminization, parthenogenesis, male killing (MK) and cytoplasmic incompatibility. In butterflies, some effects especially MK and sperm-egg incompatibility are well established. All these effects skew the sex ratio towards female and subsequently favor the vertical transmission of Wolbachia. Some of the insects are also infected with multiple Wolbachia strains which may results in some complex phenomenon. In the present review the potential of Wolbachia for promoting evolutionary changes in its hosts with emphasis on recent advances in interactions of butterfly–Wolbachia is discussed. In addition to this, strain diversity of Wolbachia and its effects on various butterfly hosts are also highlighted.     

Interaction of a Wolbachia WSP-like protein with a nuclear-encoded protein of Brugia malayi

The Brugia malayi endosymbiont Wolbachia has recently been shown to be essential for its host’s survival and development. However, relatively little is known about Wolbachia proteins that interact with the filarial host and which might be important in maintaining the obligate symbiotic relationship. The Wolbachia surface proteins (WSPs) are members of the outer membrane protein family and we hypothesise that they might be involved in the Wolbachia-Brugia symbiotic relationship. Notably, immunolocalisation studies of two WSP members, WSP-0432 and WSP-0284 in B. malayi female adult worms showed that the corresponding proteins are not only present on the surface of Wolbachia but also in the host tissues, with WSP-0284 more abundant in the cuticle, hypodermis and the nuclei within the embryos. These results confirmed that WSPs might be secreted by Wolbachia into the worm’s tissue. Our present studies focus on the potential involvement of WSP-0284 in the symbiotic relationship of Wolbachia with its filarial host. We show that WSP-0284 binds specifically to B. malayi crude protein extracts. Furthermore, a fragment of the hypothetical B. malayi protein (Bm1_46455) was found to bind WSP-0284 by panning of a B. malayi cDNA library. The interaction of WSP-0284 and this protein was further confirmed by ELISA and pull-down assays. Localisation by immunoelectron microscopy within Wolbachia cells as well as in the worm’s tissues, cuticle and nuclei within embryos established that both proteins are present in similar locations within the parasite and the bacteria. Identifying such specific interactions between B. malayi and Wolbachia proteins should lead to a better understanding of the molecular basis of the filarial nematode and Wolbachia symbiosis.     

Absence of Wolbachia in Nonfilariid Nematodes

Intracellular bacteria of the genus Wolbachia are among the most abundant endosymbionts on the planet, occurring in at least two major phyla-the Arthropoda and Nematoda. Current surveys of Wolbachia distribution have found contrasting patterns within these groups. Whereas Wolbachia are widespread and occur in all three major subphyla of arthropods, with estimates placing them in at least several million arthropod species, the presence of nematodes carrying Wolbachia is currently confined to the filariids, in which they occur at appreciable frequencies. It has been hypothesized that Wolbachia entered the ancestor of modern-day filariids in a single acquisition event, and subsequently cospeciated with their filariid hosts. To further investigate this hypothesis, we examined the broader distribution of Wolbachia in nematodes using a polymerase chain reaction (PCR) assay in a diverse set of nonfilariid species. The assay consisted of three different types of PCR screens on adults of 20 secernentean nematode species (14 rhabditids, 2 strongylid parasites of vertebrates; 1 diplogasterid; 3 cephalobid relatives, 1 myolaim, and 1 filariid) and two adenophorean species (plectids). Two PCR screens were specific to the 16S rDNA and ftsZ protein coding gene of Wolbachia; and the third screen was specific to the 18S rDNA of the nematodes. Based upon our results, we conclude that Wolbachia are absent in all 21 non-filariid species encompassing all the major groups of the Secernentea and two species of Adenophorea, from which the Secernentea derived. The absence of Wolbachia in these non-filariids is consistent with the hypothesis that Wolbachia entered the nematode phylum once, in an ancestral lineage of extant filariids.     

Conservation of the Type IV Secretion System throughout Wolbachia evolution

The Type IV Secretion System (T4SS) is an efficient pathway with which bacteria can mediate the transfer of DNA and/or proteins to eukaryotic cells. In Wolbachia pipientis, a maternally inherited obligate endosymbiont of arthropods and nematodes, two operons of vir genes, virB3-B6 and virB8-D4, encoding a T4SS were previously identified and characterized at two separate genomic loci. Using the largest data set of Wolbachia strains studied so far, we show that vir gene sequence and organization are strictly conserved among 37 Wolbachia strains inducing various phenotypes such as cytoplasmic incompatibility, feminization, or oogenesis in their arthropod hosts. In sharp contrast, extensive variation of genomic sequences flanking the virB8-D4 operon suggested its distinct location among Wolbachia genomes. Long term conservation of the T4SS may imply maintenance of a functional effector translocation system in Wolbachia, thereby suggesting the importance for the T4SS in Wolbachia biology and survival inside host cells.     

Monitoring long-term evolutionary changes following Wolbachia introduction into a novel host: the Wolbachia popcorn infection in Drosophila simulans

Wolbachia may act as a biological control agent for pest management; in particular, the Wolbachia variant wMelPop (popcorn) shortens host longevity and may be useful for dengue suppression. However, long-term changes in the host and Wolbachia genomes can alter Wolbachia spread and/or host effects that suppress disease. Here, we investigate the phenotypic effects of wMelPop in a non-native host, Drosophila simulans, following artificial transinfection approximately 200 generations ago. Long-term rearing and maintenance of the bacteria were at 19°C in the original I-102 genetic background that was transinfected with the popcorn strain. The bacteria were then introgressed into three massbred backgrounds, and tetracycline was used to create uninfected sublines. The effect of wMelPop on longevity in this species appears to have changed; longevity was no longer reduced at 25°C in some nuclear backgrounds, reflecting different geographical origin, selection or drift, although the reduction was still evident for flies held at 30°C. Wolbachia influenced productivity and viability, and development time in some host backgrounds. These findings suggest that long-term attenuation of Wolbachia effects may compromise the effectiveness of this bacterium in pest control. They also emphasize the importance of host nuclear background on Wolbachia phenotypic effects.     

Antifilarial and Antibiotic Activities of Methanolic Extracts of Melaleuca cajuputi Flowers

We evaluated the activity of methanolic extracts of Melaleuca cajuputi flowers against the filarial worm Brugia pahangi and its bacterial endosymbiont Wolbachia. Anti-Wolbachia activity was measured in worms and in Aedes albopictus Aa23 cells by PCR, electron microscopy, and other biological assays. In particular, microfilarial release, worm motility, and viability were determined. M. cajuputi flower extracts were found to significantly reduce Wolbachia endosymbionts in Aa23 cells, Wolbachia surface protein, and microfilarial release, as well as the viability and motility of adult worms. Anti-Wolbachia activity was further confirmed by observation of degraded and phagocytized Wolbachia in worms treated with the flower extracts. The data provided in vitro and in vivo evidence that M. cajuputi flower extracts inhibit Wolbachia, an activity that may be exploited as an alternative strategy to treat human lymphatic filariasis.     

Macronutrients mediate the functional relationship between Drosophila and Wolbachia

Wolbachia are maternally inherited bacterial endosymbionts that naturally infect a diverse array of arthropods. They are primarily known for their manipulation of host reproductive biology, and recently, infections with Wolbachia have been proposed as a new strategy for controlling insect vectors and subsequent human-transmissible diseases. Yet, Wolbachia abundance has been shown to vary greatly between individuals and the magnitude of the effects of infection on host life-history traits and protection against infection is correlated to within-host Wolbachia abundance. It is therefore essential to better understand the factors that modulate Wolbachia abundance and effects on host fitness. Nutrition is known to be one of the most important mediators of host–symbiont interactions. Here, we used nutritional geometry to quantify the role of macronutrients on insect–Wolbachia relationships in Drosophila melanogaster. Our results show fundamental interactions between diet composition, host diet selection, Wolbachia abundance and effects on host lifespan and fecundity. The results and methods described here open a new avenue in the study of insect–Wolbachia relationships and are of general interest to numerous research disciplines, ranging from nutrition and life-history theory to public health.     

Unravelling the Wolbachia evolutionary role: the reprogramming of the host genomic imprinting

Environmental factors can induce significant epigenetic changes that may also be inherited by future generations. The maternally inherited symbiont of arthropods Wolbachia pipientis is an excellent candidate as an ‘environmental’ factor promoting trans-generational epigenetic changes: by establishing intimate relationships with germ-line cells, epigenetic effects of Wolbachia symbiosis would be manifested as a ‘maternal effect’, in which infection of the mother modulates the offspring phenotype. In the leafhopper Zyginidia pullula, Wolbachia feminizes genetic males, leaving them as intersexes. With the exception of male chitinous structures that are present in the last abdominal segment, feminized males display phenotypic features that are typical of females. These include ovaries that range from a typical histological architecture to an altered structure. Methylation-sensitive random amplification of polymorphic DNA profiles show that they possess a female genomic imprint. On the other hand, some rare feminized males bear testes instead of ovaries. These specimens possess a Wolbachia density approximately four orders of magnitude lower than feminized males with ovaries and maintain a male genome—methylation pattern. Our results indicate that Wolbachia infection disrupts male imprinting, which dramatically influences the expression of genes involved in sex differentiation and development, and the alteration occurs only if Wolbachia exceeds a density threshold. Thus, a new Wolbachia''s role as an environmental evolutionary force, inducing epigenetic trans-generational changes, should now be considered.     

The incidence of bacterial endosymbionts in terrestrial arthropods

Intracellular endosymbiotic bacteria are found in many terrestrial arthropods and have a profound influence on host biology. A basic question about these symbionts is why they infect the hosts that they do, but estimating symbiont incidence (the proportion of potential host species that are actually infected) is complicated by dynamic or low prevalence infections. We develop a maximum-likelihood approach to estimating incidence, and testing hypotheses about its variation. We apply our method to a database of screens for bacterial symbionts, containing more than 3600 distinct arthropod species and more than 150 000 individual arthropods. After accounting for sampling bias, we estimate that 52% (CIs: 48–57) of arthropod species are infected with Wolbachia, 24% (CIs: 20–42) with Rickettsia and 13% (CIs: 13–55) with Cardinium. We then show that these differences stem from the significantly reduced incidence of Rickettsia and Cardinium in most hexapod orders, which might be explained by evolutionary differences in the arthropod immune response. Finally, we test the prediction that symbiont incidence should be higher in speciose host clades. But while some groups do show a trend for more infection in species-rich families, the correlations are generally weak and inconsistent. These results argue against a major role for parasitic symbionts in driving arthropod diversification.     

Effects of Doxycycline on gene expression in Wolbachia and Brugia malayi adult female worms in vivo

Background

Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi.

Methods

Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles.

Results and discussion

Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms) and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%). In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes). Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton) were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion).

Conclusions

Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron-transport, metabolism, anti-oxidants, and others with unknown functions had increased expression signals after doxycycline treatment. These results suggest that female worms are able to compensate in part for the loss of Wolbachia so that they can survive, albeit without reproductive capacity. This study of doxycycline induced changes in gene expression has provided new clues regarding the symbiotic relationship between Wolbachia and B. malayi.     

The endosymbiont Wolbachia increases insulin/IGF-like signalling in Drosophila

Insulin/IGF-like signalling (IIS) is an evolutionarily conserved pathway that has diverse functions in multi-cellular organisms. Mutations that reduce IIS can have pleiotropic effects on growth, development, metabolic homeostasis, fecundity, stress resistance and lifespan. IIS is also modified by extrinsic factors. For instance, in the fruitfly Drosophila melanogaster, both nutrition and stress can alter the activity of the pathway. Here, we test experimentally the hypothesis that a widespread endosymbiont of arthropods, Wolbachia pipientis, can alter the degree to which mutations in genes encoding IIS components affect IIS and its resultant phenotypes. Wolbachia infection, which is widespread in D. melanogaster in nature and has been estimated to infect 30 per cent of strains in the Bloomington stock centre, can affect broad aspects of insect physiology, particularly traits associated with reproduction. We measured a range of IIS-related phenotypes in flies ubiquitously mutant for IIS in the presence and absence of Wolbachia. We show that removal of Wolbachia further reduces IIS and hence enhances the mutant phenotypes, suggesting that Wolbachia normally acts to increase insulin signalling. This effect of Wolbachia infection on IIS could have an evolutionary explanation, and has some implications for studies of IIS in Drosophila and other organisms that harbour endosymbionts.     

Iron Necessity: The Secret of Wolbachia's Success?

The bacterium Wolbachia (order Rickettsiales) is probably the world''s most successful vertically-transmitted symbiont, distributed among a staggering 40% of terrestrial arthropod species. Wolbachia has great potential in vector control due to its ability to manipulate its hosts'' reproduction and to impede the replication and dissemination of arboviruses and other pathogens within haematophagous arthropods. In addition, the unexpected presence of Wolbachia in filarial nematodes of medical and veterinary importance has provided an opportunity to target the adult worms of Wuchereria bancrofti, Onchocerca volvulus, and Dirofilaria immitis with safe drugs such as doxycycline. A striking feature of Wolbachia is its phenotypic plasticity between (and sometimes within) hosts, which may be underpinned by its ability to integrate itself into several key processes within eukaryotic cells: oxidative stress, autophagy, and apoptosis. Importantly, despite significant differences in the genomes of arthropod and filarial Wolbachia strains, these nexuses appear to lie on a continuum in different hosts. Here, we consider how iron metabolism may represent a fundamental aspect of host homeostasis that is impacted by Wolbachia infection, connecting disparate pathways ranging from the provision of haem and ATP to programmed cell death, aging, and the recycling of intracellular resources. Depending on how Wolbachia and host cells interact across networks that depend on iron, the gradient between parasitism and mutualism may shift dynamically in some systems, or alternatively, stabilise on one or the other end of the spectrum.     

Addicted? Reduced host resistance in populations with defensive symbionts

Heritable symbionts that protect their hosts from pathogens have been described in a wide range of insect species. By reducing the incidence or severity of infection, these symbionts have the potential to reduce the strength of selection on genes in the insect genome that increase resistance. Therefore, the presence of such symbionts may slow down the evolution of resistance. Here we investigated this idea by exposing Drosophila melanogaster populations to infection with the pathogenic Drosophila C virus (DCV) in the presence or absence of Wolbachia, a heritable symbiont of arthropods that confers protection against viruses. After nine generations of selection, we found that resistance to DCV had increased in all populations. However, in the presence of Wolbachia the resistant allele of pastrel—a gene that has a major effect on resistance to DCV—was at a lower frequency than in the symbiont-free populations. This finding suggests that defensive symbionts have the potential to hamper the evolution of insect resistance genes, potentially leading to a state of evolutionary addiction where the genetically susceptible insect host mostly relies on its symbiont to fight pathogens.     

Wolbachia Infections in the Cimicidae: Museum Specimens as an Untapped Resource for Endosymbiont Surveys

Wolbachia spp. are obligate maternally inherited endosymbiotic bacteria that infect diverse arthropods and filarial nematodes. Previous microscopic and molecular studies have identified Wolbachia in several bed bug species (Cimicidae), but little is known about how widespread Wolbachia infections are among the Cimicidae. Because cimicids of non-medical importance are not commonly collected, we hypothesized that preserved museum specimens could be assayed for Wolbachia infections. For the screening of museum specimens, we designed a set of primers that specifically amplify small diagnostic fragments (130 to 240 bp) of the Wolbachia 16S rRNA gene. Using these and other previously published primers, we screened 39 cimicid species (spanning 16 genera and all 6 recognized subfamilies) and 2 species of the sister family Polyctenidae for Wolbachia infections using museum and wild-caught material. Amplified fragments were sequenced to confirm that our primers were amplifying Wolbachia DNA. We identified 10 infections, 8 of which were previously undescribed. Infections in the F supergroup were common in the subfamily Cimicinae, while infections in the A supergroup were identified in the subfamilies Afrocimicinae and Haematosiphoninae. Even though specimens were degraded, we detected infections in over 23% of cimicid species. Our results indicate that Wolbachia infections may be common among cimicids and that archived museum material is a useful untapped resource for invertebrate endosymbiont surveys. The new screening primers listed in this report will be useful for other researchers conducting Wolbachia surveys with specimens with less-than-optimum DNA quality.     

Lateral transfers of insertion sequences between Wolbachia,Cardinium and Rickettsia bacterial endosymbionts

Various bacteria live exclusively within arthropod cells and collectively act as an important driver of arthropod evolutionary ecology. Whereas rampant intra-generic DNA transfers were recently shown to have a pivotal role in the evolution of the most common of these endosymbionts, Wolbachia, the present study show that inter-generic DNA transfers also commonly take place, constituting a potent source of rapid genomic change. Bioinformatic, molecular and phylogenetic data provide evidence that a selfish genetic element, the insertion sequence ISRpe1, is widespread in the Wolbachia, Cardinium and Rickettsia endosymbionts and experiences recent (and likely ongoing) transfers over long evolutionary distances. Although many ISRpe1 copies were clearly expanding and leading to rapid endosymbiont diversification, degraded copies are also frequently found, constituting an unusual genomic fossil record suggestive of ancient ISRpe1 expansions. Overall, the present data highlight how ecological connections within the arthropod intracellular environment facilitate lateral DNA transfers between distantly related bacterial lineages.