How can I isolate live Wolbachia endosymbionts from Drosophila

How can I isolate live Wolbachia endosymbionts from Drosophila

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I am interested to culture Wolbachia bacteria in cell line.

You can see this protocol for Wolbachia isolation:

See this protocol for infecting cell lines with Wolbachia.

In this study the authors have established a cell line from a Wolbachia infected tissue of Aedes albopictus. You can have a look at that too.

Behavior of Wolbachia Endosymbionts From Drosophila Simulans in Drosophila Serrata, A Novel Host

Many species harbor the incompatibility‐inducing microbe Wolbachia, a maternally inherited endoparasite that causes reduced egg hatch in crosses between infected males and uninfected females. Infected females are immune to this effect, which gives them a relative fitness advantage that results in the spread of the infection. The strength of incompatibility, fitness deficits associated with the infection, and transmission rate from mother to offspring largely determine the rate and extent of spread of Wolbachia in a population. We transferred Wolbachia from Drosophila simulans to Drosophila serrata, a novel host, and compared parameter estimates with those from three naturally occurring Drosophila‐Wolbachia associations believed to be of different ages. Transfected D. serrata showed strong incompatibility, low transmission efficiency, and an associated fitness deficit, and they would probably be unable to spread in nature. The comparisons generally supported the predicted evolution of a host‐Wolbachia association. The parameters peculiar to any given host‐Wolbachia association may determine whether the microbial strain can spread in that host.


André C. Pimentel , Cássia S. Cesar , Marcos Martins and Rodrigo Cogni *

Wolbachia is a maternally transmitted bacterium that lives inside arthropod cells. Historically, it was viewed primarily as a parasite that manipulates host reproduction, but more recently it was discovered that Wolbachia can also protect Drosophila species against infection by RNA viruses. Combined with Wolbachia’s ability to invade insect populations due to reproductive manipulations, this provides a way to modify mosquito populations to prevent them transmitting viruses like dengue. In this review, we discuss the main advances in the field since Wolbachia’s antiviral effect was discovered 12 years ago, identifying current research gaps and potential future developments. We discuss that the antiviral effect works against a broad range of RNA viruses and depends on the Wolbachia lineage. We describe what is known about the mechanisms behind viral protection, and that recent studies suggest two possible mechanisms: activation of host immunity or competition with virus for cellular resources. We also discuss how association with Wolbachia may influence the evolution of virus defense on the insect host genome. Finally, we investigate whether the antiviral effect occurs in wild insect populations and its ecological relevance as a major antiviral component in insects.


Sample collection

SBPH individuals were collected from rice plants at 17 locations in China and Japan during the summers (May to September) of 2010–2018 (Fig. 1, left panel Additional file 1: Table S1). We haphazardly collected about 60–100 adult female individuals at each location. To avoid sampling siblings, we collected only one SBPH per host plant and selected host plants that were at least 1 m apart. All samples were preserved in 100% ethanol and stored at − 20 °C until DNA extraction.

Sampling localities (left) and infection frequencies (right) of Wolbachia in natural populations of SBPH across 17 locations. The numbers in the location map indicate the numbers of SBPH detected. Positions of the infection frequency bars correspond to the latitude of the population. The locations and dates of collection are given in Additional file 1: Table S1

16S rRNA amplicon sequencing

For each of the 17 locations, three female adults were pooled to provide a biological replicate and three biological replicates were established per location. Total genomic DNA was extracted with a DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocols. A two-step PCR approach recommended by Illumina [37] was performed to generate amplicon libraries. Briefly, the PCR amplification of the bacterial 16S rRNA genes involved universal primer sets 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR products were purified on a 2% agarose gel, and extracted with an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). The Illumina sequencing adapters and sample-specific barcodes were added to the purified PCR products with a second PCR using the TruePrep Index Kit V3 for Illumina (Vazyme, Nanjing, China). Final PCR products were purified with Hieff NGS DNA selection Beads (YEASEN, Shanghai, China), and equalized and normalized using the dsDNA HS assay kit for Qubit (YEASEN, Shanghai, China). Samples were quantified and pooled in equimolar ratio using a Qubit 4 Fluorometer (Invitrogen, Waltham, MA, USA) and then were submitted to Majorbio Bio-Pharm Technology Company Limited (Shanghai, China) for high-throughput sequencing on an Illumina MiSeq PE300.

After sequencing, raw fastq files were demultiplexed, quality-filtered by Trimmomatic, and merged by FLASH [38] ( OTUs were clustered with 97% similarity cutoff using UPARSE [39] (version 7.1, and sequences were then phylogenetically assigned to taxonomic classifications using an RDP classifier [40] ( To normalize sequencing depth, the samples were rarefied to 34135 sequences (the lowest coverage sample) to ensure a random subset of OTUs for all samples.

Mitochondrial COI gene PCR

In SPBH, no significant differentiation among populations exists for nuclear genes but mitochondrial genes that are passed down from mother are differentiated [29]. To determine the degree of genetic differentiation, 20 to 46 female adults were haphazardly selected from each population (Fig. 1, left panel) for mitochondrial COI gene amplifications and sequencing according to Sun et al. [29]. The PCR products were sent to Tsingke Biological Technology Company (China) for sequencing.

Diagnostic PCR

To measure infection frequencies of Wolbachia, an additional eight to 46 female adults were haphazardly selected from each population. The specific primers [41] are listed in Additional file 1: Table S2. DNA extraction and PCR were done as described above. Positive controls (known sample with Wolbachia) and blank controls were also run. PCR products of 599 bp size were run on 1.0% agarose gels stained with ethidium bromide at 150 volts and visualized by GenoSens 1860 (Clinx, Shanghai, China). The number of samples showing bright DNA bands compared with the DL 2000 DNA mark (Tsingke, China) was used to calculate the infection rate.

Transcriptome analyses

To investigate the effects of Wolbachia infection on the SBPH transcriptome, we compared Wolbachia-free and Wolbachia-infected SBPH strains. The uninfected strain was obtained by treating the infected strain with tetracycline for 10 generations according to the method of Guo et al. [42]. Briefly, approximately 30 abdomens of SBPH as a biological replicate were dissected from 3-day-adults of both Wolbachia-infected and Wolbachia-free females. The female abdomens contain a large quantity of fat body and blood cells which are the basis of innate immunity. Total RNA was extracted from three biological replicates using TRIzol Reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. RNA purity was measured with a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured with a Qubit® RNA Assay Kit in a Qubit® 2.0 Fluorometer (Life Technologies, CA, USA). Finally, RNA was pooled for Illumina MiSeq sequencing (BGI, Wuhan, China) according to a standard protocol [43].

The sequencing generated 6.6 Gb per biological replicate. Clean reads were obtained by removing reads with adaptors, poly-N, and having a low quality. Gene expression levels were estimated by RSEM software package [44] ( Immune-related genes of SBPH were obtained from Zhu et al. [45], which were generated by alignments with immune genes of D. melanogaster, A. gambiae, Aedes aegypti, and Culex quinquefasciatus by using BLASTX [46]. In addition, sequences were annotated to the KO database with the KEGG Automatic Annotation Server.

Statistical analyses

Bray–Curtis dissimilarity metrics among all samples were constructed using in QIIME [47] ( and were visualized with a principal coordinate analysis (PCoA). The difference of microbial communities among the populations was calculated by ADONIS. The population genetic differentiation value (FST) was calculated in Arlequin 3.5 [48]. The annual mean temperatures (Bio1) and the annual mean precipitation (Bio12) of the 17 locations were obtained from DIVA-GIS 7.5.0 [49] (, which is a geographic information system for the analysis of species distribution data. A structural equation model (SEM) [50] was used to estimate the relative contributions of FST, Bio1, Bio12, latitude, and longitude (Additional file 1: Table S3 Table S4 Table S1) on microbial community structure with communities based on Bray–Curtis dissimilarity metrics. The SEM tests were performed in the R “SEM” package (, and the path diagram for the SEM tests is shown in Fig. 4. As non-normal distribution of variables may compromise SEM analyses results, we also undertook Mantel tests using the Spearman method with 1000 permutations to determine the associations between microbial community structure variation and the five aforementioned factors.

The relative abundance of a given phylogenetic group was estimated by examining the number of reads of that group for each population. In order to analyze the evenness and richness of the microbial community, we calculated several α diversity indexes including the Sods, Shannon, Simpson, Ace, Chao, and Coverage indexes. Spearman’s rank correlations were calculated between the proportion of Wolbachia and the α diversity indexes (Shannon indexes and Simpson indexes) of the populations. The significance of differences in read proportions of bacterial 16S rRNA genes at the genus level was assessed by Mann–Whitney U tests. The significance of differences in α diversity indexes between Wolbachia-infected and -uninfected populations was calculated by a t test. All statistical analyses were carried out in R 3.5.2 [51].

Probabilistic features recognition for the OTU distribution

Components of collective ecological and biological systems presented an obvious probabilistic similarity in their aggregation, in which only several species made up a relatively high share of the whole sample, while most species accounted for much less. By looking into our datasets, we noted that the abundance data of OTUs explicitly met this property. Therefore, the power-law function that satisfied the mathematical characterization of such distribution behavior was considered as an appropriate function to recognize the probability distribution features of OTUs. Given the type of power-law function, the abundance had the probability density function (pdf):

where x’ was the threshold that ensured a robust fitting for the power-law distribution. We probabilistically characterized the distribution of abundance of OTUs by calculating the exceedance probability distribution function [52] that was given by:

where ε was the scale exponent of power-law distribution underlying the statistical patterns of data considered. This scale factor implied the property of mean and variance of data: when ε ≤ 2, the mean and variance were both infinite when 2 < ε < 3, the mean existed, while the variance was still infinite and when ε ≥ 3, both mean and variance existed. Additionally, ( fleft(frac> ight) ) was introduced to give a general formulation for the homogeneity function. The probabilistic features for the OTU distribution for each population were given in Fig. 2.

Exceedance probability distribution function of OTU abundance for each population. A power-law function is used as the model to estimate the pdf of abundances. Population codes are given in Additional file 1: Table S1

To assess the microbial community variations between populations in terms of probabilistic distributions of OTUs, we calculated the Kullback–Leibler divergence (KL divergence) by using the R package “LaplacesDemon” ( on/index.html). Probability density functions of OTUs used as the arguments for KL divergence calculation function were computed by using the R package “histogram” ( The KL divergence was used as a surrogate index of microbial community structure and was also used for SEM and Mantel tests to analyze the relationships between microbial community structure and five putative predictor variables as mentioned above.

A Wolbachia-associated fitness benefit depends on genetic background in Drosophila simulans

The α-proteobacteria Wolbachia infect a number of insect species and influence host reproduction to favour the spread of infected females through a population. The fitness effect of this infection is important in understanding the spread and maintenance of Wolbachia within and among host populations. However, a full elucidation of fitness effect requires careful control of host genetic background. Here, I transferred a single clone of Wolbachia (the wHa strain) into three genetically distinct isofemale lines of the fly Drosophila simulans using microinjection methodology. These lines carried one of the three described mitochondrial haplogroups (siI, siII or siIII) and differ in nuclear genome as well. Population cage assays showed that wHa-infected siIII flies enjoyed a dramatic fitness benefit compared to uninfected siIII. In contrast, wHa did not affect the fitness of siI or siII flies. This study points to the importance of host-by-symbiont interaction terms that may play an important role in organismal-fitness.


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Many interesting bacteria form intimate, obligate relationships with eukaryotes. These bacteria include endosymbionts and obligate intracellular pathogens. These microbes can be difficult to research as they cannot be cultured, easily manipulated, or genetically transformed. Therefore, genomics techniques have significantly advanced the study of these organisms. Because of the obvious potential health impacts, many techniques and tools have been developed to research such bacteria that interact with humans. For example, the Ribo-Zero rRNA removal kit for human/mouse/rat (Epicentre, Madison, WI, USA) can facilitate transcriptome analysis of intracellular pathogens of humans. However, for non-mammalian systems including bacteria/vector systems, a void still exists. Previously, the MICROBEnrich insect/C. elegans module (Ambion, Austin, TX, USA) was used for this purpose, but it is no longer available. Therefore, we sought to investigate if Epicentre’s Ribo-Zero rRNA removal kit designed for humans and rodents would efficiently remove rRNA from insect samples. Drosophila ananassae is a fruit fly that can be naturally infected with a Wolbachia endosymbiont [1, 2]. In addition, some Wolbachia-colonized lines have an entire Wolbachia genome transferred to a fly nuclear chromosome [3]. Transcripts from nuclear Wolbachia transfers (nuwts) are of particular interest, as they can be used to elucidate functions for the nuwts. Previously, we identified 28 nuwt transcripts (

2% of the Wolbachia genome) in adult flies, albeit at low levels [3]. Since these transcripts might not be polyadenylated, poly-A enrichment of total RNA is not a suitable technique for obtaining mRNA prior to RNASeq. Therefore, Ribo-Zero was tested as a suitable alternative.

RNA isolation, mRNA enrichment, and transcriptome sequencing

Total RNA was isolated with TRIzol, as previously described [3], from 50–85 freshly laid eggs of wild-type D. ananassae In(3R)A (Stock No. 14024–0371.13) that is infected with the Wolbachia endosymbiont w Ana [2] and from a tetracycline-treated (Wolbachia-cured) line of this fly. Both lines are tested regularly by fluorescence in situ hybridization to confirm the presence or absence of a Wolbachia infection, respectively. The samples were enriched for mRNA using the Ribo-Zero rRNA removal kit for human/mouse/rat (Epicentre, Madison, WI, USA), following the manufacturer’s protocol.

Assessment of rRNA removal by microfluidics

Comparison of the original and Ribo-Zero-subtracted samples on a Bioanalyzer (Agilent, Santa Clara, CA, USA) illustrates the efficient removal of the majority of the rRNA (Figure 1) and the loss of >97% of the RNA, as assessed by integrating the area under the rRNA peaks. In D. ananassae, as well as in many insects, the 28S rRNA is naturally cleaved resulting in two peaks that are unusually close together on the Bioanalyzer when compared to other eukaryotic or bacterial rRNA samples. Importantly, the Ribo-Zero subtraction efficiently removed both halves of the cleaved rRNA.

Bioanalyzer analysis of Ribo-Zero-subtracted RNA. The subtraction of rRNA using the Ribo-Zero human/mouse/rat reagents was tested on total RNA from Drosophila ananassae with (panels A and C) and without (panels B and D) the presence of its Wolbachia endosymbiont. The total RNA prior to Ribo-Zero subtraction (panels A and B) is compared to RNA after Ribo-Zero subtraction (panels C and D). The starting amount of RNA prior to subtraction for panels C and D was equivalent to the amount shown in panels A and B.

Assessment of rRNA removal by transcriptome sequencing

While the Bioanalyzer is highly sensitive at detecting intact rRNA, degraded rRNA can go undetected. Therefore, we also sought to examine RNASeq results to ensure even subtraction across the rRNA. Paired-end libraries (Illumina, San Diego, CA, USA) were constructed using the two pools of RNA for the cured line (Figure 1B and 1D) using the standard protocol starting immediately after the poly-A selection step. Half of a channel of 72-bp reads was obtained on a GAIIx (Illumina, San Diego, CA, USA) for each library. The sequencing reads were mapped against the reference D. ananassae assembly, using the default parameters for BWA [4], yielding 15.8 million and 15.0 million mapped reads from total RNA and the Ribo-Zero-subtracted sample, respectively. The reads for each 100 kbp window across the genome are plotted, comparing the subtracted sample from the total RNA (Figure 2). In this subtraction, and unlike the subtraction with the RNA from uncured specimens, the Bioanalyzer revealed that only 98% of rRNA was removed. Therefore, and as expected, a significant amount of rRNA is still present, as illustrated by the red dots representing fragments with at least a portion of an rRNA. However, the results are consistent with removal of >90% of the rRNA with a shift leftward of the majority of 100 kbp fragments due to enrichment for mRNA and separation of the rRNA from the mRNA by approximately one order of magnitude.

Comparison of total RNA and Ribo-Zero-subtracted RNA from Drosophila ananassae. The reads from both samples were mapped with BWA against the D. ananassae scaffolds. Each scaffold was then computationally divided into 100 kbp fragments and the number of reads mapping to the fragment were counted. When scaffolds were <100 kbp, the entire scaffold was counted. The last fragment of each scaffold was always <100 kbp. Fragments containing annotated rRNA are shifted right of the diagonal (gray line) due to decreased representation in the Ribo-Zero-treated RNA. Meanwhile, fragments without rRNA are shifted leftward of the diagonal (gray line) because of their increased abundance relative to the entire sequenced population in the subtracted samples.

While the overall trend is clear, specific points may not reflect this trend. For example, the 100 kbp region containing the 18S rRNA is the red point in the upper right corner that appears unshifted from the diagonal. In the total RNA, 2,936,794 reads mapped to the 18S rRNA fragment (41,477-43,516 bp on scaffold 13163 GenBank CH902719.1) in the subtracted sample 980,094 reads (66%) were sequenced (Figure 3A). This would be sufficient to offset the point from diagonal except that an additional

3 million reads map in this 100 kbp fragment and outside the 18S rRNA. These reads prevent the full shift of this point relative to the diagonal that would be realized should only the 18S rRNA be present in this fragment.

Coverage of the 18S rRNA and actin genes. The coverage of reads across the 18S rRNA (Panel A) and actin (Panel C) genes was determined by mpileup in samtools and compared between total RNA (solid line) and Ribo-Zero-subtracted RNA (dashed line). For the 18S rRNA, the Ribo-Zero-subtracted sample contained 66% fewer rRNA reads when compared to the total RNA sample. When the 18S rRNA results are normalized relative to actin, a 95% reduction in the rRNA is seen (Panel B). The 18S rRNA is only partially sequenced in the reference genome with a gap in the scaffold to the immediate right of this region.

While there is a 66% difference in the raw number of 18S rRNA reads between the two samples (Figure 3A), this does not fully capture the Ribo-Zero subtraction. Subtraction of rRNA means that significantly more reads are obtained from the remaining RNA species in the subtracted sample, as seen in the 6.2-fold increase in signal for actin in the subtracted sample (Figure 3C). When the rRNA results for each sample are normalized to the number of reads in actin, a 95% reduction of rRNA is observed (Figure 3B). This is consistent with the 98% reduction determined by integrating the area under the peak on the Bioanalyzer.

Not only does the subtraction increase the signal for genes as shown above with actin, it also increases the number of genes that can be analyzed. While 8,888 transcripts had at least a single read mapping in the subtracted sample, only 7,629 transcripts had a single read mapping in the unsubtracted sample. Yet, a single read is not very informative when examining differential expression, and instead, a minimum number of reads/transcript may be required for a differential expression analysis. Standards for this minimum have not been established to our knowledge. But if one required 100 reads/transcript, the subtracted sample would have 3,677 transcripts that could be analyzed while the unsubtracted sample would only have 1,047 transcripts.

Detection of transcripts arising from bacteria-to-animal lateral gene transfer

While the rRNA was sufficiently removed from the studied samples, only a few reads (19 and 20 reads from the total RNA and the Ribo-Zero-subtracted RNA, respectively) arose from transcripts of nuwts as identified by mapping with BWA [4] against the wRi reference genome [5] (Table 1). No transcripts were identified with more than one mapped read after duplicate removal. This low abundance of reads is not sufficient for analysis of the nuwt transcriptome. There was no overlap in the reads found between the two samples with the exception of reads that likely arose from non-Wolbachia bacterial contaminants on the surface of the eggs, further suggesting the stochastic detection of these low abundance transcripts.

This low abundance of transcription mirrors previous findings that transcription of nuwts is low [3]. In previous work, nuwts were found to be 10 4 -10 7 times less abundant than actin in adult flies [3], which is consistent with the RNASeq results presented here for eggs. This level of transcription may or may not be biologically relevant. Important tissue-, condition-, and/or stage-specific transcription cannot be ruled out. However the tissue, condition, and/or stage that should be examined are not immediately obvious in the absence of a phenotype.

Mechanisms of symbiosis with Wolbachia

Wolbachia are widespread bacteria in nature. They live in symbiosis with an amazing diversity of arthropod species, and are also hosted by parasitic filarial nematodes. These bacteria have the amazing peculiarity of being transmitted by the host female germ line to the offspring, like mitochondria. As Wolbachia can affect the biology of their hosts, which can be disease vectors (for instance, mosquitos or human parasites), they are of utmost biomedical relevance. Filariasis is a highly debilitating vector-borne disease that affects over 120 million people in tropical areas. It is caused by parasitic nematodes that live in mutualism with Wolbachia. Without their symbionts, these worms become sterile and quickly die. Therefore, Wolbachia is a promising drug target. We study the cellular and molecular mechanisms underlying the symbiosis with Wolbachia in nematodes and insects. To this aim, we develop new techniques of observation and investigation, especially for filarial nematodes.

Outstanding questions:
We use a multispecies approach to address key issues:

  1. What are the transmission mechanisms of Wolbachia, within an organism (from the zygote to the adult germline), and within an insect population?
  2. Why are Wolbachia required for the survival and fertility of the filarial species they colonize?
  3. How do Wolbachia subvert the host cell machinery to establish and maintain their intracellular lifestyle?

Models we use:
To address these questions, we use the natural hosts of Wolbachia as experimental models, such as Brugia malayi (the causative agent of elephantiasis) and other filarial nematodes species, as well as the Drosophila fly and the Culex pipens mosquito.

Wolbachia control stem cell behavior and stimulate germline proliferation in filarial nematodes

Vincent Foray*, Mercedes M.Perez-Jiménez*, Nour Fattouh, Frédéric Landmann

Developmental Cell, accepted.

Contact our team

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Members of the team

IL-4 receptor dependent expansion of lung CD169+ macrophages in microfilaria-driven inflammation

Frédéric Fercoq, Estelle Remion, Stefan J Frohberger, Nathaly Vallarino-Lhermitte, Achim Hoerauf, John Le Quesne, Frédéric Landmann, Marc P Hübner, Leo[. ]

Discovery of short-course antiwolbachial quinazolines for elimination of filarial worm infections

Malina A Bakowski, Roohollah Kazem Shiroodi, Renhe Liu, Jason Olejniczak, Baiyuan Yang, Kerstin Gagaring, Hui Guo, Pamela M White, Laura[. ]

Sci Transl Med 11(491):eaav3523 Pubmed

Wolbachia endosymbionts subvert the endoplasmic reticulum to acquire host membranes without triggering ER stress.

Fattouh N, Cazevieille C, Landmann F.

PLoS Negl Trop Dis. 13(3):e0007218. Pubmed

The Wolbachia Endosymbionts.

The cellular phenotype of cytoplasmic incompatibility in Culex pipiens in the light of cidB diversity

Manon Bonneau, Frédéric Landmann, Pierrick Labbé, Fabienne Justy, Mylène Weill, Mathieu Sicard

Wolbachia Control Stem Cell Behavior and Stimulate Germline Proliferation in Filarial Nematodes.

Foray V, Pérez-Jiménez MM, Fattouh N, Landmann F.

Non-centrosomal epidermal microtubules act in parallel to LET-502/ROCK to promote C. elegans elongation.

Quintin S, Wang S, Pontabry J, Bender A, Robin F, Hyenne V, Landmann F, Gally C, Oegema K, Labouesse M.

Drosophila protamine-like Mst35Ba and Mst35Bb are required for proper sperm nuclear morphology but are dispensable for male fertility.

Tirmarche S, Kimura S, Sapey-Triomphe L, Sullivan W, Landmann F, Loppin B.

Co-evolution between an endosymbiont and its nematode host: Wolbachia asymmetric posterior localization and AP polarity establishment.

Landmann F, Foster JM, Michalski ML, Slatko BE, Sullivan W.

PLoS Negl Trop Dis. 28:e3096. Pubmed

Absence of Wolbachia endobacteria in the human parasitic nematode Dracunculus medinensis and two related Dracunculus species infecting wildlife.

Foster JM, Landmann F, Ford L, Johnston KL, Elsasser SC, Schulte-Hostedde AI, Taylor MJ, Slatko BE.

Interdomain lateral gene transfer of an essential ferrochelatase gene in human parasitic nematodes.

Wu B, Novelli J, Jiang D, Dailey HA, Landmann F, Ford L, Taylor MJ, Carlow CK, Kumar S, Foster JM,[. ]

Proc Natl Acad Sci U S A. 110(19):7748-53. Pubmed

Efficient in vitro RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, Brugia malayi.

Landmann F, Foster JM, Slatko BE, Sullivan W.

A cell-based screen reveals that the albendazole metabolite, albendazole sulfone, targets Wolbachia.

Serbus LR, Landmann F, Bray WM, White PM, Ruybal J, Lokey RS, Debec A, Sullivan W.

A new type F Wolbachia from Splendidofilariinae (Onchocercidae) supports the recent emergence of this supergroup.

Lefoulon E, Gavotte L, Junker K, Barbuto M, Uni S, Landmann F, Laaksonen S, Saari S, Nikander S, de Souza[. ]

Int J Parasitol. 42(11):1025-36. Pubmed

Both asymmetric mitotic segregation and cell-to-cell invasion are required for stable germline transmission of Wolbachia in filarial nematodes.

Landmann F, Bain O, Martin C, Uni S, Taylor MJ, Sullivan W.

A tension-induced mechanotransduction pathway promotes epithelial morphogenesis.

Zhang H, Landmann F, Zahreddine H, Rodriguez D, Koch M, Labouesse M.

New insights into the evolution of Wolbachia infections in filarial nematodes inferred from a large range of screened species.

Ferri E, Bain O, Barbuto M, Martin C, Lo N, Uni S, Landmann F, Baccei SG, Guerrero R, de Souza[. ]

Anti-filarial activity of antibiotic therapy is due to extensive apoptosis after Wolbachia depletion from filarial nematodes.

Landmann F, Voronin D, Sullivan W, Taylor MJ.

Asymmetric Wolbachia segregation during early Brugia malayi embryogenesis determines its distribution in adult host tissues.

Landmann F, Foster JM, Slatko B, Sullivan W.

PLoS Negl Trop Dis. 4(7):e758. Pubmed

Wolbachia-mediated cytoplasmic incompatibility is associated with impaired histone deposition in the male pronucleus.

Landmann F, Orsi GA, Loppin B, Sullivan W.

The genetics and cell biology of Wolbachia-host interactions.

Serbus LR, Casper-Lindley C, Landmann F, Sullivan W.

Wolbachia and filarial nematodes.

Filarial parasites cause neglected tropical diseases (filiariases) that affect more than 120 million people. Filiriases are debilitating diseases and include elephantiasis (lymphatic filariasis) and the african river blindness (Onchocerciasis). The Wolbachia endosymbionts are required for proper fertility and survival of adult filarial nematodes. Moreover, they participate in the pathology caused by the filarial nematodes by triggering inflammatory reactions when released from dead larvae and are the cause of blindness in Onchocerciasis. Wolbachia are now a major drug target to fight filarial diseases because the current anti-parasitic treatments do not kill adult worms. This is a serious problem because worms can live more than a decade in their host and the females release hundreds of larvae every day.

During the embryogenesis of Brugia malayi (one of the causative agents of elephantiasis) Wolbachia segregate asymmetrically to reach a subset of hypodermal precursors. In juvenile adult females, the endosymbionts present in the hypodermis show an ovarian tropism and colonize the germline.
We are interested in the cellular and molecular characterization of these complex transmission mechanisms.

Cytoplasmic Incompatibility.

Fruit fly species and mosquitoes, among many other species of insects, can be natural Wolbachia hosts. In both cases, Wolbachia parasites are not essential for their host survival/fertility, but they manipulate the host reproduction machinery to their own advantage in order to ensure their transmission to the next generation. Indeed, Wolbachia bacteria use a strategy called Cytoplasmic Incompatibility (CI) to spread in a given insect population. CI is a complex process that leads to the death of uninfected progeny resulting from non-infected females fertilized by infected males (a dead end for maternally transmitted bacteria). Therefore, infected females and their infected progeny end up with a selective advantage. Specifically, fertilization of non-infected eggs by sperm from a Wolbachia-infected male no longer leads to normal embryo development. As early as the first zygotic division, the paternal chromatin shows remodelling defects and fails to condense and segregate properly, leading to aneuploid or haploid development, which is lethal in flies and mosquitoes.
We are interested in CI molecular bases in order to understand how Wolbachia can modify the sperm characteristics and how their presence also in infected eggs restores the sperm-egg compatibility to allow normal development after fertilization by infected males.

The Wolbachia pandemic | Symbionts spread rapidly across highly diverged flies

Wolbachia are perhaps the most prevalent bacterial symbionts on earth. Of the millions of insect species, Wolbachia are estimated to infect up to half of them. These bacteria are renowned for the effects they exert on their hosts, which can often be quite dramatic. Some Wolbachia strains are highly pathogenic for instance, the wMelPop, or “popcorn” strain causes the cells of their fly hosts to rupture, leading to an early death. In mosquitoes, the infection can render their proboscis bendy and completely useless for blood-sucking. However as Wolbachia can only live within the cells of host insects, it’s in their best interests to keep their hosts alive.

Wolbachia are transmitted from mother to offspring, and so they often employ strategies that increase the number of females that are infected with the bacteria. For instance, they can turn male offspring into females or even induce parthenogenesis, allowing females to produce more females without the need for males at all. Some strains can also provide their hosts with direct benefits, such as increasing the number of eggs they lay, or protecting them against pathogens. Please see our recent blog post for a general overview of Wolbachia.

These features enable Wolbachia infections to spread throughout populations of insects. In some cases, these Wolbachia “pandemics” can occur extremely rapidly. In the 1990s, Australian Drosophila simulans flies were mostly Wolbachia-free, with a low proportion being infected with a strain called wAu. By 2012, almost all flies across the eastern coast of Australia were now infected with the Wolbachia strain wRi, with the wAu strain nowhere to be seen. The wRi strain induces cytoplasmic incompatibility, which reduces the fertility of uninfected females that mate with males infected with Wolbachia. But infected females can produce viable offspring regardless of who they mate with, giving them the upper hand in reproduction.

Wolbachia strains have also been introduced deliberately into mosquito populations hijacking this same mechanism of cytoplasmic incompatibility. The Wolbachia strain wMel, which provides mosquitoes with anti-viral protection was introduced into Cairns, Australia, and now most mosquitoes in the area are less capable of transmitting dengue. But in this case Wolbachia were transferred artificially, by injecting Wolbachia into mosquito eggs in the laboratory. How is it that Wolbachia became so widespread in insects if it’s transmitted from a mother to her offspring?

A recent study from a collaboration between PEARG and Californian researchers documents the rapid spread of Wolbachia (wRi-like) into eight different species of Drosophila flies around the world. Some of these species are quite distant relatives, with their common ancestor being at least 10 million years in the past. But the Wolbachia they are infected with are much more closely related, diverging no more than 30 thousand years ago. Wolbachia host shifts were previously thought to occur very rarely, but in the case of these Drosophila species and wRi Wolbachia have jumped from fly species to fly species on multiple occasions and in a very short period of a few thousand years.

In nature, Wolbachia can be transferred from one species to another in two main ways. The first is though inter-breeding, but Wolbachia can only be transferred in this way when two species are closely related. This new study also finds evidence of horizontal transfer between distantly related Drosophila. In this case, the mechanism behind the host shift is unclear, but there are a few ways in which Wolbachia can jump to another species. The bacteria could hitch-hike via mites or parasitoids. Wolbachia might even be transferred through cannibalism and predation. However this was expected to be an extremely rare process occurring only every few million years. In contrast to the rapid spread of Wolbachia between species, the authors find no evidence of horizontal transfer occurring within species. This means that each infected individual of a species likely originated from a single infected female.

Another surprising finding of this research is that despite their genetic similarity, the wRi-like Wolbachia strains have different effects in different hosts. In Drosophila anomalata, Wolbachia induces cytoplasmic incompatibility but its counterpart in Drosophila suzukii has no effect on fly reproduction. This means that Wolbachia can still invade a species even without this reproductive advantage, though Wolbachia must induce some beneficial effects for the infection to persist.

wRi-like Wolbachia strains seem to be particularly good at spreading throughout populations once they are introduced to a novel host, and could therefore have applications for disease and pest control if they are introduced artificially into other insects. The wRi strain from Drosophila simulans has already been introduced into the dengue vector mosquito Aedes aegypti as a potential biological control agent. Although this strain doesn’t block dengue transmission very effectively, wRi has minimal negative effects on the mosquito and induces very strong cytoplasmic incompatibility. This could make it very useful for introducing anti-viral genes into areas where dengue is endemic, and could even be used for suppressing or eradicating mosquito populations through cytoplasmic incompatibility.

Editors note: this story has now also been reviewed in The California Aggie and Current Biology


Stocks and fly rearing

The eight wild-type lines used for this experiment were selected from the Drosophila Genetic Reference Panel (Mackay et al. 2012). These lines are: RAL149, RAL306, RAL321, RAL365, RAL790, RAL853, RAL879, and RAL897. All of these strains have standard chromosome arrangements. These lines are naturally infected with Wolbachia pipientis (Huang et al. 2014). Genomic analysis indicates that the colonizing Wolbachia strain is a wMel-like strain (Richardson et al. 2012). wMel has been shown to induce cytoplasmic incompatibility, though the magnitude of the effect varies among studies (Bourtzis et al. 1996 Poinsot et al. 1998 Mcgraw et al. 2002 Reynolds and Hoffmann 2002 Yamada et al. 2007). We created Wolbachia-free versions of these eight strains. To cure the strains of Wolbachia infection, we raised flies on tetracycline-containing media for two consecutive generations. These flies were raised in 8 ounce (oz) bottles with a standard cornmeal/molasses media containing tetracycline (dissolved in ethanol) to a final concentration of 0.25 mg/ml media. After the second generation of tetracycline treatment, these strains (denoted, for example, RAL149 w - ) were raised on standard media for more than five generations before the experiment described below began.

We used doubly-marked strains for our estimation of recombination rate. The markers used to measure recombination on the X chromosome were yellow (y 1 ) and vermilion (v 1 ) (Bloomington Drosophila Stock Center #1509), which are 33 cM apart (Lindsley and Grell 1967). We integrated this doubly-marked X chromosome into the wild-type isogenic Samarkand genetic background (Lyman et al. 1996) this line abbreviated hereafter as ‘y v’. The markers on the chromosome 3R were ebony (e 4 ) and rough (ro 1 ) (Bloomington Drosophila Stock Center #496), which are 20.4 cM apart (Lindsley and Grell 1967) this line is abbreviated hereafter as ‘e ro.’ These markers and strains have been used extensively in our lab to estimate recombination frequency (Jackson et al. 2015 Singh et al. 2015 Hunter et al. 2016a Hunter et al. 2016b). Both of the marker strains have a standard chromosome arrangement.

Wolbachia screen

Immediately prior to conducting these experiments, we confirmed the presence of Wolbachia infection in the standard RAL lines (denoted, for example, RAL149 w + ) and the absence of Wolbachia in the tetracycline-treated lines using a PCR-based assay with Wolbachia-specific primers. Four adult females were used per line to test for Wolbachia infection. Briefly, DNA was extracted from each female using a standard squish prep (Gloor and Engels 1992). Each fly was crushed with a motorized pestle and subsequently immersed in a buffered solution (10 mM Tris-Cl pH 8.2, 1 mM EDTA, 25 mM NaCl, 200 μg/ml proteinase K). This was incubated at 37° for 30 min and then at 95° for 2 min to inactivate the proteinase K. We used Wolbachia-specific primers wspF and wspR (Jeyaprakash and Hoy 2000) to test for presence/absence of Wolbachia infection.

Amplifying conditions were as follows: 94°/3 min, 12 cycles of 94°/30 sec, 65°/30 sec, 72°/60 sec with the annealing temperature reduced by 1.0 degrees per cycle, followed by 25 cycles of 94°/30 sec, 55°/30 sec, 72°/60 sec. We included a final extension of 72°/7 min. All PCR reactions were 10 μl, and each contained 5 μl Qiagen 2X PCR Master- Mix, 0.25 μl of each 20 mM primer, 3.5 μl H2O, and 1 μl genomic DNA. All four tested females from each of the eight Wolbachia-infected lines showed evidence of Wolbachia infection using this assay, while none of the females from the tetracycline-treated lines showed any evidence for Wolbachia infection.

Experimental crosses

To assay recombination rate variation in the experimental lines, we used a classic two-step backcrossing scheme. All crosses were executed at 25° with a 12:12 hr light:dark cycle on standard media using virgin females aged roughly 24 hr. For the first cross, ten virgin females from each experimental evolution line were crossed to ten e ro (or y v) males in 8 oz bottles. Males and females were allowed to mate for five days, after which all adults were cleared from the bottles. F1 females resulting from this cross are doubly heterozygous these females are the individuals in which recombination is occurring. To uncover these recombination events we backcrossed F1 females to doubly-marked males. For this second cross, twenty heterozygous virgin females were collected and backcrossed to twenty doubly-marked males in 8 oz bottles. Males and females were allowed to mate for five days, after which all adults were cleared from the bottles. After eighteen days, BC1 progeny were collected and scored for sex and for visible phenotypes. Recombinant progeny were then identified as having only one visible marker (e+ or +ro in the case of crosses involving the e ro double mutant, or y+ or +v in the crosses involving the y v double mutant). Ten to fifteen replicate (second) crosses were set up for each strain. For each replicate, recombination rate was estimated by taking the ratio of recombinant progeny to the total number of progeny. Double crossovers cannot be recovered with this assay, so our estimates of recombination frequency are likely to be biased downward slightly.

Statistical analyses

All statistics were conducted using JMPPro v13.0. To test for factors associated with variation in recombination fraction, we used a generalized linear model with a binomial distribution and logit link function on the proportion of progeny that is recombinant. We treated each offspring as a realization of a binomial process (either recombinant or nonrecombinant), summarized the data for a given vial by the number of recombinants and the number of trials (total number of progeny per vial), and tested for an effect of line and Wolbachia status plus the interaction of line and Wolbachia status. We note that ‘line’ incorporates known differences between host genotypes and potential unknown differences in Wolbachia genotype. This logistic regression approach thus takes the total number of observations per vial into account (giving more weight to vials with more progeny, where the estimation of recombination is likely to be more accurate). The full model is as follows: Y represents the proportion of progeny that is recombinant, μ represents the mean of regression, and ε represents the error. L denotes strain, W denotes Wolbachia infection status, and LxW denotes the interaction of line and Wolbachia infection status. All of these are modeled as fixed effects.

To test for potential differences between the two different intervals surveyed, we employed a similar logistic approach. The full model is as follows: Y represents the proportion of progeny that is recombinant, μ represents the mean of regression, and ε represents the error. L denotes strain, W denotes Wolbachia infection status, and I denotes interval surveyed. There are four interaction terms: LxW denotes the interaction of line and Wolbachia infection status, LxI is the interaction between line and interval, IxW is the interaction between interval and Wolbachia infection status, and LxWxI is the interaction among line, Wolbachia infection status and interval. All of these are modeled as fixed effects.

We note that whether ‘line’ could in principle be modeled as a fixed effect or a random effect, the choice of which to employ depends on the specific question that one is posing. If we wished to use the lines in the current experiment to say something about populations of D. melanogaster broadly speaking, then should model ‘line’ as a random effect. If instead we merely wish to interrogate this specific set of lines, not representative of any population, then modeling ‘line’ as a fixed effect is statistically appropriate. Our results speak to this set of lines alone, and we thus model line as a fixed effect. We have modeled ‘line’ as a fixed effect for similar questions in previous work (Hunter and Singh 2014 Hunter et al. 2016a Hunter et al. 2016b Kohl and Singh 2018).

To test for factors associated with variation in the sex ratio, we used the same generalized linear model framework with a binomial distribution and logit link function on the proportion of total number of progeny that is male. For each interval, the model is the same as is described above, except that Y represents the proportion of progeny that is male. We note that this analysis is independent of recombination frequency.

To test for factors associated with variation in reproductive output, we used an ANOVA framework. The ANOVA followed the form of Y = μ + L + W + ϵ, for each interval assayed where Y is reproductive output, μ is the overall mean, L is the fixed effect of line, W is the fixed effect of Wolbachia status, and ϵ is the residual.

Availability of data and material

Strains are available upon request. Raw data have been deposited into Dryad (doi:10.5061/dryad.16dt35k).


The Lepidoptera have emerged as important models in the study of the genetic and functional basis of the reproductive manipulations heritable endosymbionts employ, particularly with regard to Wolbachia bacteria. The results of this cumulative work is suggestive of the role of endosymbionts in the evolution of host sex determination itself. We have no doubt Lepidopteran endosymbiont research will continue to highlight the omnipresence and importance of Wolbachia but we suggest that more attention should now be given to the presence and interaction of other heritable endosymbionts Lepidoptera carry. Metagenomic approaches enable an unbiased view of the microbial community residing within moths and butterflies, while comparative endosymbiont genomics may illuminate the genetic mechanisms underlying the phenotypes endosymbionts induce in their host. Finally, given the importance of Lepidoptera as key indicators of climate change and the growing numbers of species listed as endangered, the study of heritable microbial endosymbiont in the Lepidoptera should transition from being a pure science filled with interesting curiosities, to a necessity that will contribute to the preservation of natural biodiversity and inform conservation management.

Watch the video: Bruzzese: Wolbachia induced cytoplasmic incompatibility in Rhagoletis (August 2022).