Large Genomes Assembly Using MAPREDUCE Framework

Knowing the genome sequence of an organism is the essential step toward understanding its genomic and genetic characteristics. Currently, whole genome shotgun (WGS) sequencing is the most widely used genome sequencing technique to determine the entire DNA sequence of an organism. Recent advances in next-generation sequencing (NGS) techniques have enabled biologists to generate large DNA sequences in a high-throughput and low-cost way. However, the assembly of NGS reads faces significant challenges due to short reads and an enormously high volume of data. Despite recent progress in genome assembly, current NGS assemblers cannot generate high-quality results or efficiently handle large genomes with billions of reads. In this research, we proposed a new Genome Assembler based on MapReduce (GAMR), which tackles both limitations. GAMR is based on a bi-directed de Bruijn graph and implemented using the MapReduce framework. We designed a distributed algorithm for each step in GAMR, making it scalable in assembling large-scale genomes. We also proposed novel gap-filling algorithms to improve assembly results to achieve higher accuracy and more extended continuity. We evaluated the assembly performance of GAMR using benchmark data and compared it against other NGS assemblers. We also demonstrated the scalability of GAMR by using it to assemble loblolly pine (~22Gbp). The results showed that GAMR finished the assembly much faster and with a much lower requirement of computing resources

Genome Mining Actinobacteria: Eliciting the Production of Natural Products

Antimicrobial resistance is an imminent threat that is expected to kill 10 million people per year by 2050. Natural products have been a major source of antimicrobial compounds and encompass a chemical space far greater than synthetic chemistry can provide and have evolved over the ages to have biological activities. The natural products produced by Streptomyces have provided us with two-thirds of the antibiotics currently used, as well as chemotherapeutics, antifungals, and immunosuppressants. In recent years, the drug discovery pipeline from Streptomyces has run dry, largely because laboratory culture conditions lack their natural stimuli, resulting in the rediscovery of the same natural products. However, with the advent of modern genomics, we now realize that the genomes of Streptomyces have the have a greater capacity for natural product production than what we have observed, which providing us with the hope of new drug leads. My doctoral research focused on two aims. First is the concept of eliciting Streptomyces to produce novel natural products; in other words, what triggers the production of natural products. Additional perspectives from ecology, evolution, and regulation evoked the idea that microbe-microbe interactions could be the key. Microscopic observations of the interactions between Streptomyces and yeast suggested that physical contact is essential for elicitation. Genomics, transcriptomics, and proteomics showed that 31% of the silent biosynthetic gene clusters are activated, notably an antifungal polyene cluster, as well as a suite of enzymes capable of digesting the cell wall of yeast. Arguably, Streptomyces can prey on yeast. The differential regulation of a homologous polyene gene clusters further suggested that natural product production is triggered by different ecological needs. Second, genome mining provides insight into the genetic potential of Actinobacteria to produce natural products via the identification of their gene clusters and is a proven method that aids in drug discovery. Here, I sequenced the genome of a rare Streptomonospora isolate and identified the novel persiamycin gene cluster and its associated product. Moreover, genome mining was applied to publicly available Streptomyces genomes, which resulted in the identification of two new gene clusters that produce the antibiotic komodoquinone B. KEYWORDS: Streptomyces, Actinobacteria, secondary metabolism, natural products, genome miningMikrobilääkeresistenssi on välitön uhka, joka uhkaa tappaa vuosittain 10 miljoonaa ihmistä vuoteen 2050 mennessä. Luonnontuotteet ovat olleet merkittävä mikrobilääkkeiden lähde. Lisäksi luonnontuotteet kattavat paljon laajemman kemiallisen alueen kuin synteettinen kemia voi tarjota, ja ne ovat aikojen kuluessa kehittyneet niin, että niillä on biologisia vaikutuksia. Streptomykeetti-organismin tuottamat luonnontuotteet ovat tuottaneet kaksi kolmasosaa käytössä olevista antibiooteista sekä kemoterapeuttisia aineita, sienilääkkeitä, immunosuppressantteja ja matolääkkeitä. Viime vuosina Streptomykeetti-bakteerin lääkkeiden kehittämiskanava on kuitenkin kuivunut. Nykyaikaisen genomiikan myötä olemme kuitenkin nyt ymmärtäneet, että Streptomykeeteillä on potentiaalia tuottaa vielä löytämättömiä luonnontuotteita, jotka antavat meille toivoa uusista lääkkeistä, erityisesti antibiooteista. Väitöstutkimuksellani oli kaksi tavoitetta. Ensimmäinen tarkoitus oli saada Streptomykeetit tuottamaan uusia luonnontuotteita; toisin sanoen tutkia sitä, mikä saa Streptomykeetin tuottamaan luonnontuotteita. Ekologian, evoluution ja säätelyn lisänäkökulmat herättivät ajatuksen, että mikrobien väliset vuorovaikutus voisivat olla avainasemassa. Mikroskooppiset havainnot Streptomykeetin ja hiivan välisestä vuorovaikutuksesta osoittivat, että fyysinen kontakti oli välttämätön yhdisteiden tuoton aktivoinnissa. Genomiikka, transkriptomiikka ja proteomiikka osoittivat, että jopa 31 prosenttia hiljaisista biosynteettisistä geeniryhmistä aktivoitui, samoin kuin joukko entsyymejä, jotka kykenevät pilkkomaan hiivan soluseinää. Tämä osoitti, että Streptomykeetit pystyvät saalistamaan hiivasoluja. Homologisten geeniryhmien erilainen säätely viittaa lisäksi siihen, että luonnontuotteiden tuotanto perustuu erilaisiin ekologisiin tarpeisiin. Toisena tavoitteena oli käyttää genomien louhintaa selvittääksemme Streptomykeetin geneettisestä potentiaalista tuottaa luonnontuotteita. Tässä työssä selvitimme harvinaisen Streptomonaspora bakteerin genomin ja tunnistin uuden persiamysiini-yhdisteen biosynteesireitin. Tämän lisäksi löysimme genomin louhinnan avulla kaksi uutta biosynteettistä geeniryhmää julkisista tietokannoista, joiden osoitimme olevan vastuussa komodokinoni B antibiootin tuotannosta. ASIASANAT: Streptomykeetit, Aktinobakteerit, sekundaarinen aineenvaihdunta, luonnonyhdisteet, genomin louhint

Variable recombination dynamics during the emergence, transmission and ‘disarming’ of a multidrug-resistant pneumococcal clone

Background: Pneumococcal β-lactam resistance was first detected in Iceland in the late 1980s, and subsequently peaked at almost 25% of clinical isolates in the mid-1990s largely due to the spread of the internationally-disseminated multidrug-resistant PMEN2 (or Spain6B-2) clone of Streptococcus pneumoniae. Results: Whole genome sequencing of an international collection of 189 isolates estimated that PMEN2 emerged around the late 1960s, developing resistance through multiple homologous recombinations and the acquisition of a Tn5253-type integrative and conjugative element (ICE). Two distinct clades entered Iceland in the 1980s, one of which had acquired a macrolide resistance cassette and was estimated to have risen sharply in its prevalence by coalescent analysis. Transmission within the island appeared to mainly emanate from Reykjavík and the Southern Peninsular, with evolution of the bacteria effectively clonal, mainly due to a prophage disrupting a gene necessary for genetic transformation in many isolates. A subsequent decline in PMEN2’s prevalence in Iceland coincided with a nationwide campaign that reduced dispensing of antibiotics to children in an attempt to limit its spread. Specific mutations causing inactivation or loss of ICE-borne resistance genes were identified from the genome sequences of isolates that reverted to drug susceptible phenotypes around this time. Phylogenetic analysis revealed some of these occurred on multiple occasions in parallel, suggesting they may have been at least temporarily advantageous. However, alteration of ‘core’ sequences associated with resistance was precluded by the absence of any substantial homologous recombination events. Conclusions: PMEN2’s clonal evolution was successful over the short-term in a limited geographical region, but its inability to alter major antigens or ‘core’ gene sequences associated with resistance may have prevented persistence over longer timespans

Visualization Tools for Comparative Genomics applied to Convergent Evolution in Ash Trees

Assembly and analysis of whole genomes is now a routine part of genetic research, but effective tools for the visualization of whole genomes and their alignments are few. Here we present two approaches to allow such visualizations to be done in an efficient and user-friendly manner. These allow researchers to spot problems and patterns in their data and present them effectively. First, FluentDNA is developed to tackle single full genome visualization and assembly tasks by representing nucleotides as colored pixels in a zooming interface. This enables users to identify features without relying on algorithmic annotation. FluentDNA also supports visualizing pairwise alignments of wellassembled whole genomes from chromosome to nucleotide resolution. Second, Pantograph is developed to tackle the problem of visualizing variation among large numbers of whole genome sequences. This uses a graph genome approach, which addresses many of the technical challenges of whole genome multiple sequence alignments by representing aligned sequences as nodes which can be shared by many individuals. Pantograph is capable of scaling to thousands of individuals and is applied to SARS and A. thaliana pangenomes. Alongside the development of these new genomics tools, comparative genomic research was undertaken on worldwide species of ash trees. I assembled 13 ash genomes and used FluentDNA to quality check the results and discovered contaminants and a mitochondrial integration. I annotated protein coding genes in 28 ash assemblies and aligned their gene families. Using phylogenetic analysis, I identified gene duplications that likely occurred in an ancient whole genome duplication shared by all ash species. I examined the fate of these duplicated genes, showing that losses are concentrated in a subset of gene families more often than predicted by a null model simulation. I conclude that convergent evolution has occurred in the loss and retention of duplicated genes in different ash species.BBSRC BB/S004661/

BMC Biol

BackgroundPneumococcal \uce\ub2-lactam resistance was first detected in Iceland in the late 1980s, and subsequently peaked at almost 25% of clinical isolates in the mid-1990s largely due to the spread of the internationally-disseminated multidrug-resistant PMEN2 (or Spain6B-2) clone of Streptococcus pneumoniae.ResultsWhole genome sequencing of an international collection of 189 isolates estimated that PMEN2 emerged around the late 1960s, developing resistance through multiple homologous recombinations and the acquisition of a Tn5253-type integrative and conjugative element (ICE). Two distinct clades entered Iceland in the 1980s, one of which had acquired a macrolide resistance cassette and was estimated to have risen sharply in its prevalence by coalescent analysis. Transmission within the island appeared to mainly emanate from Reykjav\uc3\uadk and the Southern Peninsular, with evolution of the bacteria effectively clonal, mainly due to a prophage disrupting a gene necessary for genetic transformation in many isolates. A subsequent decline in PMEN2\ue2\u20ac\u2122s prevalence in Iceland coincided with a nationwide campaign that reduced dispensing of antibiotics to children in an attempt to limit its spread. Specific mutations causing inactivation or loss of ICE-borne resistance genes were identified from the genome sequences of isolates that reverted to drug susceptible phenotypes around this time. Phylogenetic analysis revealed some of these occurred on multiple occasions in parallel, suggesting they may have been at least temporarily advantageous. However, alteration of \ue2\u20ac\u2dccore\ue2\u20ac\u2122 sequences associated with resistance was precluded by the absence of any substantial homologous recombination events.ConclusionsPMEN2\ue2\u20ac\u2122s clonal evolution was successful over the short-term in a limited geographical region, but its inability to alter major antigens or \ue2\u20ac\u2dccore\ue2\u20ac\u2122 gene sequences associated with resistance may have prevented persistence over longer timespans.098051/Wellcome Trust/United Kingdo

Conservation of different mechanisms of Hox cluster regulation within chordates

[eng] In this thesis we have covered the importance of finding underlying conservation events to better understand the regulatory mechanisms of important development orchestrators like the Hox cluster. As an example of these non-evident conservation, we have shown two cases, as described below. The first case studied, after developing a software able to detect homologous long noncoding RNAs by means of microsynteny analyses, is the conservation of Hotairm1 in Chordata. For assessing the homology of this lncRNA, first we had to identify the lncRNA fraction within the B. lanceolatum transcriptome. With a reliable lincRNA dataset, we used our pipeline, LincOFinder, to identify orthologs between human and amphioxus through microsynteny. After the identification of Hotairm1 as one of the lincRNAs with conserved microsynteny, we used Xenopus as a proxy to analyse the homologies in the expression and the function. We had to proceed this way due to the difficulties associated with the inhibition of genes in B. lanceolatum, and the unavailability of expression patterns for Hotairm1 in the bibliography. After we successfully characterised Hotairm1 expression in amphioxus and Xenopus, we injected morpholino oligonucleotides to target and inhibit the splicing of Hotairm1 to promote an isoform imbalance. Through the phenotype obtained and the performing of qPCRs, we were able to deduct the mechanism of Hotairm1 and successfully relate this mechanism with the one described in human cells. With all the data obtained we were able to strongly suggest that the amphioxus Hotairm1 is homologous to the Xenopus and human Hotairm1, thus being conserved in most of the lineages within chordates. The second case studied was the conservation of the regulation of the Hox cluster mediated by Cdx. When analysing the B. floridae knockouts of Cdx and Pdx obtained using the TALEN technique, we found a severe phenotype of the developing larvae in Cdx-/- and a mild phenotype in Pdx-/-. The Cdx-/- phenotype consisted in the disruption of posterior gut development, as well as an underdevelopment of the postanal tail, coupled with a non-opening anus. When looking at changes in the expression of the Hox cluster in this Cdx-/- embryos, we found collinear misregulation of the expressed Hox genes, with the most anterior Hox cluster genes upregulated, and the most posterior ones downregulated. This is very similar to findings seen in triple morpholino knockdowns of the Cdx genes in Xenopus, indicating that in both, Xenopus and amphioxus, Cdx is regulating the Hox cluster through a homologous mechanism

Elucidating the mating system of Phaffia rhodozyma, an astaxanthin-producing yeast with biotechnological potential

In fungi belonging to the phylum Basidiomycota, sexual identity is usually determined by two genetically unlinked MAT loci, one named PR locus, which encodes one or more pheromone receptors (Ste3) and pheromone precursors (Mfa), and the other, named HD locus, that comprehends at least one pair of divergently transcribed genes encoding homeodomain transcription factors (HD1 and HD2). The two MAT loci work as two distinct mating compatibility check points. Most basidiomycete species are heterothallic, meaning that sexual reproduction requires mating between two sexually compatible individuals harboring different alleles at both MAT loci. However, some species are known to be homothallic, one individual can complete the sexual cycle without mating with a genetically distinct partner. While the molecular underpinnings of the heterothallic life cycles of several basidiomycete model species have been dissected in detail, much less is known concerning the molecular basis for homothallism. The general aim of this research was to study the molecular mechanisms of sexual reproduction in fungi, specifically those governing the homothallic life cycle of P. rhodozyma. Six MAT genes were found in P. rhodozyma, organized in two MAT loci, most likely located on different chromosomes. The PR locus was shown to be composed of two clusters, at approximately 5 kb from one another, each encoding one STE3 gene and one MFA gene, while the HD locus encompassed two divergently transcribed homeodomain transcription factors genes, HD1 and HD2. Functional genetic analysis was performed by targeted gene deletion of the MAT elements found in P. rhodozyma and the results allowed the proposal of a molecular model controlling the homothallic sexual behavior of P. rhodozyma. In this model (i) each pheromone interacts with the pheromone receptor of the other cluster (Mfa1 activates Ste3-2 while Mfa2 activates Ste3-1); since neither pheromone receptor is required per se for sporulation they seem to be functionally redundant; and (ii) both homeodomain proteins appear to work together to regulate genes required for sexual development. Comparison of the MAT regions of additional Phaffia species and of other representatives within the order Cystofilobasidiales, indicate that transitions to homothallism probably occurred several times independently. Furthermore, it revealed a particularly dynamic pattern of MAT gene evolution, with the generation of new receptors within each genus and exceptionally large numbers of mature pheromones encoded in the genomes of some of the species. In conclusion, this work allowed for the first time the elucidation of the basic molecular mechanisms governing the homothallic life cycle of a basidiomycete. At the same time the genetic manipulation of the MAT genes of P. rhodozyma also allowed the generation of preferably outcrossing strains, which may be potentially useful for further improvement of this yeast as an industrial organism by way of selective breeding

Interactions of insertion sequences targetting integron associated recombination sites

Bacteria of genus Pseudomonas are known for crossing ecological barriers and establishing in clinical settings. Their ability to withstand antibiotic selection in the clinical environment is largely due to interactions between different mobile genetic elements (MGEs) and the presence of multidrug resistance integrons (MRIs). It is difficult to predict resistance gene maintenance within a bacterial population, as the source of these genes is unknown, and the biological processes governing their flow is difficult to quantify in vivo. This study explores the IS1111-attC subgroup of insertion sequences as a model for this process in Pseudomonads. In this study, IS1111-attC elements were found to be overrepresented within non-clinical Pseudomonas isolates relative to clinical Pseudomonads, as well as an enteric outgroup. The observed IS1111-attC distribution suggests that all instances of IS1111-attC elements in class 1 integrons represent recent invasions of attC sites occurring when class 1 integrons were present in the same cells as chromosomal integrons. Target site preferences and transposition mechanisms of the IS1111-attC elements distribution patterns were investigated using in vitro and in vivo models. These elements were shown to specifically recognize the attC recombination sites of integrons in binding assays and to specifically target the attC in mobility assays. Factors affecting the rate of movement between environmental and clinical Pseudomonads were also examined. Significantly, the IS1111-attC transposase binds preferentially to the single strand forms of the top strand of the attC site, rather than the bottom strand attC site which is the target of the integron integrase. This is the first evidence for IS1111-attC mobility in Pseudomonas cells occurring via a similar mechanism to integron gene mobilization, illustrating a way for these elements both to move between chromosomal and plasmid borne integrons, and to facilitate interactions between them

Next generation sequencing for studying viruses and RNA silencing-based antiviral defense in crop plants

The main objectives of this work have been to use next generation sequencing (NGS) and develop bioinformatics tools for plant virus diagnostics and genome reconstruction as well as for investigation of RNA silencing-based antiviral defense. In virus-infected plants, the host Dicer-like (DCL) enzymes process viral double-stranded RNAs into 21-24 nucleotide (nt) short interfering RNAs (siRNAs) which can potentially associate with Argonaute (AGO) proteins and guide the resulting RNA-induce silencing complexes (RISCs) to target complementary viral RNA for post-transcriptional silencing and, in the case of DNA viruses, complementary viral DNA for transcriptional silencing. In the pioneering work, Kreuze et al. (2009) have demonstrated that an RNA virus genome can be reconstructed from multiple siRNA contigs generated by the short sequencing read assembler Velvet. In this PhD study, we developed a bioinformatics pipeline to analyze viral siRNA populations in various model and crop plants experimentally infected with known viruses and naturally infected with unknown viruses. First, we developed a bioinformatics tool (MISIS) to view and analyze maps of small RNAs derived from viruses and genomic loci that generate multiple small RNAs (Seguin et al. 2014b). Using MISIS, we discovered that viral siRNAs cover the entire genomes of RNA and DNA viruses as well as viroids in both sense and antisense orientation without gaps (Aregger et al. 2012; Seguin et al. 2014a; Rajeswaran et al. 2014a, 2014b), thus allowing for de novo reconstruction of any plant virus or viroid from siRNAs. Then, we developed a de novo assembly pipeline to reconstruct complete viral genomes as single contigs of viral siRNAs, in which Velvet was used in combination with other assemblers: Metavelvet or Oases to generate contigs from viral redundant or non-redundant siRNA reads and Seqman to merge the contigs. Furthermore, we employed the mapping tool BWA and the map viewing tool IGV to verify the reconstructed genomes and identify a consensus master genome and its variants present in the virus quasispecies. The approach combining deep siRNA sequencing with the bioinformatics tools and algorithms, which enabled us to reconstruct consensus master genomes of RNA and DNA viruses, was named siRNA omics (siRomics) (Seguin et al. 2014a). We utilized siRomics to reconstruct a DNA virus and two viroids associated with an emerging grapevine red leaf disease and generate an infectious wild type genome clone of oilseed rape mosaic virus (Seguin et al. 2014a). Furthermore, siRomics was used to investigate siRNA-based antiviral defense in banana plants persistently infected with six distinct banana streak pararetroviruses (Rajeswaran et al. 2014a) and rice plants infected with rice tungro bacilliform pararetrovirus (Rajeswaran et al. 2014b). Our results revealed that multiple host DCLs generate abundant and diverse populations of 21-, 22- and 24-nt viral siRNAs that can potentially associate with multiple AGO proteins to target viral genes for post-transcriptional and transcriptional silencing. However, pararetroviruses appear to have evolved silencing evasion mechanisms such as overexpression of decoy dsRNA from a short non-coding region of the virus genome to engage the silencing machinery in massive siRNA production and thereby protect other regions of the virus genome from repressive action of viral siRNAs (Rajeswaran et al. 2014b). Furthermore, despite massive production of 24-nt siRNAs, the circular viral DNA remains unmethylated and therefore transcriptionally active, while the host genome is extensively methylated (Rajeswaran et al. 2014b). These findings shed new light at the siRNA generating machinery of economically-important crop plants. Our analysis of plant small RNAs in banana and rice revealed a novel class of highly abundant 20-nt small RNAs with 5'-terminal guanidine (5'G), which has not been identified in dicot plants. Interestingly, the 20-nt 5'G-RNA-generating pathway does not target the pararetroviruses, which correlates with silencing evasion (Rajeswaran et al. 2014a, 2014b). This thesis work is a part of the European Cooperation in Science and Technology (COST) action that aims develop an RNA-based vaccine to immunize crop plants against viral infection. Our analysis of viral siRNA profiles in various virus-infected plants allowed to identify the regions in the viral genomes that generate low-abundance siRNAs, which are the candidate regions to be targeted by RNA interference (RNAi). Our analysis of RNAi transgenic tomato plants confirmed that targeting of the low-abundance siRNA region of Tomato yellow leaf curl virus (TYLCV) by transgene-derived siRNAs renders immunity to TYLCV disease, one of the major constraints for tomato cultivation worldwide

Exploring the Role of Wolbachia Endobacteria in the Biology of Filarial Nematode Parasites

Filarial nematodes are vector borne parasitic worms that cause a variety of disfiguring and disabling diseases, including lymphatic filariasis and onchocerciasis. Many filarial species require Wolbachia endobacteria: family: Rickettsiaceae) to carry out their life cycle. Studies using antibiotics to target the endobacteria, thereby interfering with worm fertility and viability, have generated interest in using Wolbachia as an antifilarial drug target. However, the exact mechanisms underpinning this interesting mutualistic interaction are poorly understood. Wolbachia-dependence is not ubiquitous in the filarial family. Some species are able to survive in the absence of an endosymbiont. The inconsistent patterns of Wolbachia-dependence and independence seen in filarial nematodes may be explained by two hypotheses. Following infection with the endobacteria, reductive evolution could have removed redundant genes or pathways present in both partners. Thus, deletions in the worm\u27s genome would render it dependent on Wolbachia for vital gene products. Conversely, Wolbachia-dependent species could re-acquire vital genes from the endosymbiont by horizontal gene transfer, rendering the bacteria expendable. Mitochondria and Wolbachia are co-transmitted vertically from mother to offspring, therefore the mitochondrial genome: mtDNA) is particularly sensitive to evolutionary pressures exerted by the endosymbiont. Wolbachia is also thought to be closely related to the mitochondrial progenitor, so they may overlap in function: e.g., energy production). In order to address our first hypothesis, we sequenced the mitochondrial genomes of several species of Wolbachia-dependent and independent filarial nematodes in hopes of finding some degeneracy in the mtDNA of the Wolbachia-dependent species. Our studies have shown that the mtDNA of all examined species encodes the same 12 protein coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes. Despite a careful analysis, no sequence-level differences were observed between the mtDNA of infected and uninfected species. In order to address our second hypothesis, we surveyed the genomes of two Wolbachia-independent filarial species, Acanthocheilonema viteae and Onchocerca flexuosa, in search of evidence of horizontal gene transfer from Wolbachia. Many genomic fragments containing regions with high homology to Wolbachia sequences were identified. Follow-up transcriptomic and proteomic analyses in O. flexuosa have shown that Wolbachia-like sequnces are expressed at the RNA and protein levels. Imaging studies indicate that Wolbachia-like RNAs are mainly produced in tissues known to harbor Wolbachia in infected species, while a Wolbachia-like protein was found nearby but not in the same tissue. This project has produced a vast amount of data that will be useful to the filariasis research community, including the mtDNA sequences of five filarial species, genomic sequences from A. viteae and O. flexuosa, transcriptomic sequences from O. flexuosa and a survey of the O. flexuosa adult worm proteome. Our results have verified a longstanding hypothesis that the ancestor(s) of many Wolbachia-free filarial nematode species was colonized in the distant past despite the present lack of endobacteria. Future studies may prove that horizontally transferred bacterial genes are necessary for the survival of Wolbachia-free filarial worms that would otherwise require Wolbachia for reproduction and development