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Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: A streamlined genome overrun by mobile genetic elements
... The changes to the host's metabolism are especially significant [83]. Given Wolbachia's reliance on host nutrients and its limited metabolic machinery, it likely reflects a complex interplay [84]. Wolbachia's numerous amino acid transporters suggest it actively acquires specific metabolites, potentially leading the host to either compensate for altered amino acid levels or restrict metabolite availability to limit Wolbachia's access. ...
... Wolbachia's numerous amino acid transporters suggest it actively acquires specific metabolites, potentially leading the host to either compensate for altered amino acid levels or restrict metabolite availability to limit Wolbachia's access. Wolbachia's amino acid transporters likely disrupt host amino acid levels [84], impacting not only protein synthesis but also other metabolic pathways, such as purine and pyrimidine nucleotide synthesis. Lindsey et al. [80] investigated how the gene prat2, involved in purine synthesis, affects the Wolbachia-virus-host interaction. ...
... The complete genomes of Wolbachia strains infecting both arthropods and nematodes have been sequenced, and specific gene variants have been analyzed across numerous strains. Arthropodinfecting Wolbachia genomes range from 1.2 to 1.6 million base pairs (Mb), while nematode-infecting Wolbachia genomes are smaller, ranging from 0.9 to 1.1 Mb [84,89]. Arthropod Wolbachia, like other obligate intracellular bacteria, have reduced metabolic capabilities, though they can still synthesize nucleotide triphosphates like ATP [84,89,90]. ...
Dengue fever poses a significant global health challenge, particularly in tropical and subtropical regions. Current control strategies, heavily reliant on vector control, face limitations due to insecticide resistance and logistical challenges in large urban areas. This book chapter explores the innovative potential of Wolbachia bacteria as a biocontrol agent against dengue. Wolbachia infection in Aedes aegypti mosquitoes, the primary dengue vector, has demonstrated promising results in reducing both viral replication within the mosquito and onward transmission to humans. This chapter examines the complex mechanisms underlying Wolbachia’s antiviral effects, including both immune system modulation and competition for host cell resources. Furthermore, the role of gene expression changes in mediating these effects, highlighting the potential for long-term, sustainable dengue suppression, is discussed. While challenges remain in large-scale implementation and the potential for viral evolution, Wolbachia-based interventions offer a new avenue for integrated vector management and hold considerable promise for reducing the global burden of dengue fever. Gene drive technology and thorough testing, such as analyzing virus sequences, are also discussed to evaluate how well Wolbachia works.
... However, Ae. albopictus shows strong blocking when newly infected with the wMel strain [44]. Wolbachia infection in insect cell cultures reduces host cell proliferation due to the bacterium's resource demands and high replication density [45]. No doubt Wolbachia imposes a metabolic cost, leading to reduced host fitness, it can also provide significant benefits during viral infections and nutritional stress [46]. ...
... In mosquito tissues, the Wolbachia strains wAlbB and wPip (wAlbB being antiviral, wPip not) are found at higher levels than the wMel strain [55,56]. TEM analyses revealed that wMel and wAlbB are frequently surrounded by the ER membranes, possibly to utilize host-derived membrane components for their formation [45,57]. In contrast, DENV extensively modifies ER membranes for their replication [58][59][60]. ...
... Consequently, Wolbachia can produce metabolic alterations in its host, potentially disrupting DENV replication. Both DENV viruses and Wolbachia lack essential genes for making all the lipids they need to replicate [45,65]. This competition for limited lipids resources in the mosquito is a leading theory for how Wolbachia disrupts DENV infection. ...
The insufficiency of traditional dengue control approaches has led to exploring Wolbachia as a method to reduce mosquito transmission. This chapter highlights Wolbachia, a symbiotic bacterium, as an innovative mosquito control strategy. Wolbachia exhibits antiviral properties, potentially by priming the host immune system and competing for cellular resources. An important direction for controlling the dengue virus is understanding these mechanisms in Wolbachia-mediated pathogen interference. This understanding can optimize Wolbachia’s application in controlling dengue and other mosquito-borne diseases. This chapter proposes Wolbachia as a game-changing tool in the fight against dengue and other diseases, offering hope in combating this escalating global health issue.
... For example, Wolbachia is known to modulate the host cytoskeleton (Reviewed in [46]) iron [47, e.g. 48], carbohydrates [49,50], amino acids [e.g. 50,51], nucleotides [e.g. ...
... 48], carbohydrates [49,50], amino acids [e.g. 50,51], nucleotides [e.g. 52], the endoplasmic reticulum [e.g. ...
... This measure was used as a proxy as virus particles could not be counted directly. The number of infectious units (IU) was estimated using TCID 50 . Wolbachia had no statistically significant impact on the infectivity of progeny DCV (t-test, t = -1.2, ...
Viruses transmitted by arthropods pose a huge risk to human health. Wolbachia is an endosymbiotic bacterium that infects various arthropods and can block the viral replication cycle of several medically important viruses. As such, it has been successfully implemented in vector control strategies against mosquito-borne diseases, including Dengue virus. Whilst the mechanisms behind Wolbachia-mediated viral blocking are not fully characterised, it was recently shown that viruses grown in the presence of Wolbachia in some Dipteran cell cultures are less infectious than those grown in the absence of Wolbachia. Here, we investigate the breadth of this mechanism by determining if Wolbachia reduces infectivity in a different system at a different scale. To do this, we looked at Wolbachia’s impact on insect viruses from two diverse virus families within the whole organism Drosophila melanogaster. Drosophila C virus (DCV; Family Dicistroviridae) and Flock House virus (FHV; Famliy Nodaviridae) were grown in adult D. melanogaster flies with and without Wolbachia strain wMelPop. Measures of the physical characteristics, infectivity, pathogenicity, and replicative properties of progeny virus particles did not identify any impact of Wolbachia on either DCV or FHV. Therefore, there was no evidence that changes in infectivity contribute to Wolbachia-mediated viral blocking in this system. Overall, this is consistent with growing evidence that the mechanisms behind Wolbachia viral blocking are dependent on the unique tripartite interactions occurring between the host, the Wolbachia strain, and the infecting virus.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12866-024-03722-6.
... The first sequenced Wolbachia genome, strain wMel of Drosophila melanogaster, demonstrated that this genus deviates from the canonical model of streamlined genome content typical of other endosymbionts. It instead contains unusually high levels of repetitive DNA content and mobile elements such as prophage insertions, insertion sequence (IS) elements, and transposons along with large numbers of gene duplications and genetic rearrangements (Wu et al. 2004). Further, there is evidence for frequent activity and transmission of these transposable elements (TEs) in the genome (Cordaux et al. 2008). ...
... Or, these regions can transfer to different strains which allows for the acquisition of new genes, allowing for adaptation to better propagate within their hosts (Lawrence 1999;Dutta and Pan 2002). Regarding Wolbachia specifically, studies have demonstrated that lateral transfer with eukaryotes has likely occurred many times with Wolbachia, as it is common for entire Wolbachia genes or domains within genes to have homology with eukaryotic DNA (Wu et al. 2004;Bordenstein and Bordenstein 2016). Indeed, the transfers occur both ways, as many arthropod and nematode genomes also contain Wolbachia DNA (Kondo et al. 2002;Hotopp et al. 2007;Funkhouser-Jones et al. 2015;Leclercq et al. 2016;Wang et al. 2016). ...
... Genetic transfers may be aided by the abundant mobile elements within the genome, including phage WO and transposons. Indeed, the high and variable number of TEs in Wolbachia genomes has long been recognized as a useful method for differentiating strains due to their frequent movement (Wu et al. 2004;Duron et al. 2005), and they may be behind the movement of specific genes (Cooper et al. 2019) or disruption of others (Iturbe-Ormaetxe et al. 2005). In addition, the first plasmid of Wolbachia was recently described, pWCP of several populations of Culex pipiens mosquitoes, which itself contains a TE (Reveillaud et al. 2019). ...
Wolbachia are a genus of widespread bacterial endosymbionts in which some strains can hijack or manipulate arthropod host reproduction. Male killing is one such manipulation in which these maternally transmitted bacteria benefit surviving daughters in part by removing competition with the sons for scarce resources. Despite previous findings of interesting genome features of microbial sex ratio distorters, the population genomics of male-killers remain largely uncharacterized. Here, we uncover several unique features of the genome and population genomics of four Arizonan populations of a male-killing Wolbachia strain, wInn, that infects mushroom-feeding Drosophila innubila. We first compared the wInn genome to other closely related Wolbachia genomes of Drosophila hosts in terms of genome content and confirm that the wInn genome is largely similar in overall gene content to the wMel strain infecting D. melanogaster. However, it also contains many unique genes and repetitive genetic elements that indicate lateral gene transfers between wInn and non-Drosophila eukaryotes. We also find that, in line with literature precedent, genes in the Wolbachia prophage and Octomom regions are under positive selection. Of all the genes under positive selection, many also show evidence of recent horizontal transfer among Wolbachia symbiont genomes. These dynamics of selection and horizontal gene transfer across the genomes of several Wolbachia strains and diverse host species may be important underlying factors in Wolbachia’s success as a male-killer of divergent host species.
... The impact of the D. melanogaster > RML12 > Ae. aegypti host transfers on the wMel genome is not well understood. Since the initial sequencing of wMel directly from its native D. melanogaster host in 2004 [28], only one study has sequenced the wMel genome in Ae. aegypti. The study showed little variation in the sequenced genomes compared to the reference genome. ...
... The wMel genome contains high levels of repetitive DNA, mobile genetic elements and three prophage sequences -two Wolbachia prophage (phage WO) and one pyocin-like element [28]. Large expansion or movements of prophage WO have been identified in other Wolbachia genomes [31,32]. ...
... using default parameters, and these reads were excluded from downstream analysis. Single nucleotide variants were identified via mapping the remaining Illumina reads to the D. melanogaster wMel reference genome [28] (NC_002978.6) using the RedDog v1b.11 pipeline (https:// github. ...
Infection of w Mel Wolbachia in Aedes aegypti imparts two signature features that enable its application for biocontrol of dengue. First, the susceptibility of mosquitoes to viruses such as dengue and Zika is reduced. Second, a reproductive manipulation is caused that enables w Mel introgression into wild-type mosquito populations. The long-term success of this method relies, in part, on evolution of the w Mel genome not compromising the critical features that make it an attractive biocontrol tool. This study compared the w Mel Wolbachia genome at the time of initial releases and 1–7 years post-release in Cairns, Australia. Our results show the w Mel genome remains highly conserved up to 7 years post-release in gene sequence, content, synteny and structure. This work suggests the w Mel genome is stable in its new mosquito host and, therefore, provides reassurance on the potential for w Mel to deliver long-term public-health impacts.
... MGEs are not always maintained through natural selection. The genome of Wolbachia pipientis wMel, an obligate intracellular symbiont of the fruitfly Drosophila melanogaster, is highly streamlined from extensive gene loss during adaptation to its host; however, it is also overrun with MGEs 248 . Repeated population bottlenecks resulting in genetic drift and inefficient natural selection 248 likely contributes to the extensive maintenance of MGEs in this genome and those of other heritable symbionts 249 . ...
... The genome of Wolbachia pipientis wMel, an obligate intracellular symbiont of the fruitfly Drosophila melanogaster, is highly streamlined from extensive gene loss during adaptation to its host; however, it is also overrun with MGEs 248 . Repeated population bottlenecks resulting in genetic drift and inefficient natural selection 248 likely contributes to the extensive maintenance of MGEs in this genome and those of other heritable symbionts 249 . These elements may have contributed to the substantial phenotypic diversity among Wolbachia strains, fundamentally shaping Wolbachia evolution 248 . ...
... Repeated population bottlenecks resulting in genetic drift and inefficient natural selection 248 likely contributes to the extensive maintenance of MGEs in this genome and those of other heritable symbionts 249 . These elements may have contributed to the substantial phenotypic diversity among Wolbachia strains, fundamentally shaping Wolbachia evolution 248 . In this instance, MGEs are parasitic elements maintained within the population effectively by accident via transmission of Wolbachia from one host to the next. ...
Virtually all plants and animals, including humans, are home to symbiotic microorganisms. Symbiotic interactions can be neutral, harmful or have beneficial effects on the host organism. However, growing evidence suggests that microbial symbionts can evolve rapidly, resulting in drastic transitions along the parasite–mutualist continuum. In this Review, we integrate theoretical and empirical findings to discuss the mechanisms underpinning these evolutionary shifts, as well as the ecological drivers and why some host–microorganism interactions may be stuck at the end of the continuum. In addition to having biomedical consequences, understanding the dynamic life of microorganisms reveals how symbioses can shape an organism’s biology and the entire community, particularly in a changing world.
... The first sequenced genome in the genus was from supergroup A wMel strain in Drosophila melanogaster, which revealed a small 1.3 megabase (Mb) genome stunningly littered with mobile genetic elements (Wu et al., 2004). The genome sequences from wBm of Brugia malayi (Foster et al., 2005), wPip of C. pipens (Klasson et al., 2008), wRi of D. simulans (Klasson et al., 2009), and wAlbB of Aedes albopictus (Mavingui et al., 2012) followed thereafter. ...
... Wolbachia also have many repetitive regions and unusually high numbers of mobile elements presumably leading to their genome expansion (Cerveau et al., 2011;Cordaux et al., 2008;Wu et al., 2004). Genomes of nematode Wolbachia, however, lack prophage WO and harbor few mobile elements, repetitive DNA, and pseudogenes (Foster et al., 2005). ...
... The genomes of Wolbachia harbor a high frequency of transposable elements, including many insertion sequences (IS) that can occupy 10% of the bacterial genome in some cases Ling and Cordaux, 2010;Wu et al., 2004). Group II introns that are self-splicing, mobile ribozymes are also exceptionally abundant in Wolbachia relative to both endosymbiotic and free-living bacterial genomes; they can be involved in Wolbachia genomic rearrangements and extensive horizontal gene transfers . ...
The most widespread intracellular bacteria in the animal kingdom are maternally-inherited endosymbionts of the genus Wolbachia. Their prevalence in arthropods and nematodes worldwide and a stunning arsenal of parasitic and mutualistic adaptations make these bacteria a biological archetype for basic studies of symbiosis and applied outcomes for curbing human and agricultural diseases. Here, we conduct a summative, centennial analysis of living in the Wolbachia world. We synthesize literature on Wolbachia’s host range, phylogenetic diversity, genomics, cell biology, and applications to filarial, arboviral, and agricultural diseases. We also review the mobilome of Wolbachia including phage WO and its essentiality to hallmark phenotypes in arthropods. Finally, the Wolbachia system is an exemplar for discovery-based science education using biodiversity, biotechnology, and bioinformatics lessons. As we approach a century of Wolbachia research, applications, and education, the interdisciplinary science and knowledge from this symbiosis stand as a model for consolidating and teaching the integrative rules of endosymbiotic life.
... Research on Wolbachia in Drosophila melanogaster and related species has made major contributions to our understanding of Wolbachia population dynamics [17,21,26,27]. The Wolbachia endosymbiont from D. melanogaster (wMel) was the first Wolbachia strain to have its genome fully sequenced [28]. Natural populations of D. melanogaster on all continents are known to be polymorphic for Wolbachia infection [1,17,19,[29][30][31][32], but initial studies in D. melanogaster using marker loci revealed no sequence diversity in among Wolbachia isolates [17,33,34]. ...
... Moran et al. [76] also found than silent sites in Buchnera had a two-fold higher short-term evolutionary rate relative to the genome-wide estimate that includes coding sites, in contrast to what we observe in Wolbachia. The principal observation that Wolbachia has a much lower short-term evolutionary rate than Buchnera is consistent with Wolbachia having functional DNA repair pathways [28], and helps explains why Wolbachia has not undergone such extensive genome erosion as is observed in Buchnera [78]. Moreover, the different rate and pattern of mutation between Buchnera and Wolbachia genomes argues against the application of single universal ! ...
Wolbachia are maternally-inherited symbiotic bacteria commonly found in arthropods, which are able to manipulate the reproduction of their host in order to maximise their transmission. Here we use whole genome resequencing data from 290 lines of Drosophila melanogaster from North America, Europe and Africa to predict Wolbachia infection status, estimate cytoplasmic genome copy number, and reconstruct Wolbachia and mtDNA genome sequences. Complete Wolbachia and mitochondrial genomes show congruent phylogenies, consistent with strict vertical transmission through the maternal cytoplasm and imperfect transmission of Wolbachia. Bayesian phylogenetic analysis reveals that the most recent common ancestor of all Wolbachia and mitochondrial genomes in D. melanogaster dates to around 8,000 years ago. We find evidence for a recent incomplete global replacement of ancestral Wolbachia and mtDNA lineages, which is likely to be one of several similar incomplete replacement events that have occurred since the out-of-Africa migration that allowed D. melanogaster to colonize worldwide habitats.
... [63] to search for homologs of the single-copy genes in the proteobacteria database. As a control, we performed the same search using the reference wMel genome [64]. ...
... To extract genes for our phylogenetic analyses and identify homologs to known bacterial genes, we annotated our draft Smith+ genome and the public genomes of wAna from D. ananassae [69]; wAu, wHa, wRi, and wNo from D. simulans [34,70,71]; wMel from D. melanogaster [64]; wNFa, wNFe, wNLeu, and wNPa from Nomada bees [72]; wPip_Pel from Culex pipiens [36]; and wYak from D. yakuba [25] with Prokka v.1.11 [73]. ...
Bacteria that live inside the cells of insect hosts (endosymbionts) can alter the reproduction of their hosts, including the killing of male offspring (male killing, MK). MK has only been described in a few insects, but this may reflect challenges in detecting MK rather than its rarity. Here, we identify MK Wolbachia at a low frequency (around 4%) in natural populations of Drosophila pseudotakahashii. MK Wolbachia had a stable density and maternal transmission during laboratory culture, but the MK phenotype which manifested mainly at the larval stage was lost rapidly. MK Wolbachia occurred alongside a second Wolbachia strain expressing a different reproductive manipulation, cytoplasmic incompatibility (CI). A genomic analysis highlighted Wolbachia regions diverged between the 2 strains involving 17 genes, and homologs of the wmk and cif genes implicated in MK and CI were identified in the Wolbachia assembly. Doubly infected males induced CI with uninfected females but not females singly infected with CI-causing Wolbachia. A rapidly spreading dominant nuclear suppressor genetic element affecting MK was identified through backcrossing and subsequent analysis with ddRAD SNPs of the D. pseudotakahashii genome. These findings highlight the complexity of nuclear and microbial components affecting MK endosymbiont detection and dynamics in populations and the challenges of making connections between endosymbionts and the host phenotypes affected by them.
... Mitochondria have been placed in various regions of the tree of the Alphaproteobacteria. Most early studies suggested that mitochondria were most closely related to the Rickettsiales [13][14][15][16][17][18][19][20] (Rickettsiales-sister hypothesis), a group classically known for comprising intracellular parasites. This led many to believe that mitochondria evolved from parasitic alphaproteobacteria 18,21 . ...
... Thickened branches represent branch support values of >90% Shimodaira-Hasegawa approximate likelihood ratio test (SH-aLRT) and >90% UltraFast Bootstrap 2 with NNI optimization (UFBoot2 + NNI). CAT in PhyloBayes or C60 in IQ-TREE) 11,12,[14][15][16][17][18][19][20]22,23,32 . Indeed, no tractable model that accounts for compositional heterogeneity across branches and sites simultaneously is available; current branch-heterogeneous models cannot be combined with site-heterogenous models 31 , or are too computationally intensive and suffer from convergence problems 45,46 . ...
Determining the phylogenetic origin of mitochondria is key to understanding the ancestral mitochondrial symbiosis and its role in eukaryogenesis. However, the precise evolutionary relationship between mitochondria and their closest bacterial relatives remains hotly debated. The reasons include pervasive phylogenetic artefacts as well as limited protein and taxon sampling. Here we developed a new model of protein evolution that accommodates both across-site and across-branch compositional heterogeneity. We applied this site-and-branch-heterogeneous model (MAM60 + GFmix) to a considerably expanded dataset that comprises 108 mitochondrial proteins of alphaproteobacterial origin, and novel metagenome-assembled genomes from microbial mats, microbialites and sediments. The MAM60 + GFmix model fits the data much better and agrees with analyses of compositionally homogenized datasets with conventional site-heterogenous models. The consilience of evidence thus suggests that mitochondria are sister to the Alphaproteobacteria to the exclusion of MarineProteo1 and Magnetococcia. We also show that the ancestral presence of the crista-developing mitochondrial contact site and cristae organizing system (a mitofilin-domain-containing Mic60 protein) in mitochondria and the Alphaproteobacteria only supports their close relationship.
... The protein domain annotations of the assembled genomes showed 57 (4.0%) proteins in the wDi genome to contain at least one copy of an ankyrin repeat domain (Figures S3; Table S6) which is comparable to ANK proteins wMel, wRi, and wPip with about 4% of the total genes 43 . These ANK proteins of about 33 amino acids play significant role in interactions between host and symbionts 34,44 and are found abundantly in genes of WO-prophage 44 . ...
... The protein domain annotations of the assembled genomes showed 57 (4.0%) proteins in the wDi genome to contain at least one copy of an ankyrin repeat domain (Figures S3; Table S6) which is comparable to ANK proteins wMel, wRi, and wPip with about 4% of the total genes 43 . These ANK proteins of about 33 amino acids play significant role in interactions between host and symbionts 34,44 and are found abundantly in genes of WO-prophage 44 . ...
Wolbachia, a gram-negative α-proteobacterium, is an endosymbiont found in some arthropods and
nematodes. Diaphorina citri Kuwayama, the vector of ‘Candidatus Liberibacter asiaticus’ (CLas),
are naturally infected with a strain of Wolbachia (wDi), which has been shown to colocalize with the
bacteria pathogens CLas, the pathogen associated with huanglongbing (HLB) disease of citrus. The
relationship between wDi and CLas is poorly understood in part because the complete genome of
wDi has not been available. Using high-quality long-read PacBio circular consensus sequences, we
present the largest complete circular wDi genome among supergroup-B members. The assembled
circular chromosome is 1.52 megabases with 95.7% genome completeness with contamination
of 1.45%, as assessed by checkM. We identifed Insertion Sequences (ISs) and prophage genes
scattered throughout the genomes. The proteins were annotated using Pfam, eggNOG, and COG
that assigned unique domains and functions. The wDi genome was compared with previously
sequenced Wolbachia genomes using pangenome and phylogenetic analyses. The availability of a
complete circular chromosome of wDi will facilitate understanding of its role within the insect vector,
which may assist in developing tools for disease management. This information also provides a
baseline for understanding phylogenetic relationships among Wolbachia of other insect vectors.
... Phage WO is believed to be a dynamic element having a significant impact on the genomic evolution of Wolbachia (Wu et al., 2004). As in other prokaryotes, the integration and transformation of prophage are considered major sources of Wolbachia lateral gene acquisition (Bordenstein et al., 2006). ...
... A phage genome can be divided into functional units or modules (each one responsible for head or tail formation, lysis, lysogeny, and so forth), which can be mixed by segment reassortment with other phages (Hatfull, 2008). Insertion sequences are frequently found in phage WO genomes and are considered to be a major factor driving these recombinations (Wu et al., 2004;Klasson et al., 2009). The nucleotide sequence of the minor capsid gene orf7 from the wKueA1 strain of Wolbachia is chimeric, and the population genetic analysis has confirmed the occurrence of intragenic recombination events (Bordenstein and Wernegreen, 2004). ...
The phage WO was characterized in Wolbachia, a strictly intracellular bacterium causing several reproductive alterations in its arthropod hosts. This study aimed to screen the presence of Wolbachia and phage WO in 15 gall wasp species from six provinces of southern China to investigate their diversity and prevalence patterns. A high incidence of Wolbachia infection was determined in the gall wasp species, with an infection rate of 86.7% (13/15). Moreover, seven species had double or multiple infections. All Wolbachia-infected gall wasp species were found to harbor phage WO. The gall wasp species infected with a single Wolbachia strain were found to harbor a single phage WO type. On the contrary, almost all species with double or multiple Wolbachia infections harbored a high level of phage WO diversity (ranging from three to 27 types). Six horizontal transfer events of phage WO in Wolbachia were found to be associated with gall wasps, which shared identical orf7 sequences among their respective accomplices. The transfer potentially took place through gall inducers and associated inquilines infected with or without Wolbachia. Furthermore, 10 putative recombination events were identified from Andricus hakonensis and Andricus sp2, which harbored multiple phage WO types, suggesting that intragenic recombination was the important evolutionary force, which effectively promoted the high level of phage WO diversity associated with gall wasps.
... Wolbachia may rely on the host lysosome pathway for amino acids Due to its intracellular lifestyle, Wolbachia lacks many essential biosynthetic pathways, many of which are involved in amino acid production [47,48]. Therefore, Wolbachia must obtain amino acids from their hosts [47][48][49][50]. Previous studies have shown that Wolbachia is highly dependent on host proteolysis via ubiquitination and the endoplasmic reticulum-associated protein degradation (ERAD) pathway [14]. ...
... Wolbachia infection is associated with more active lipid metabolism in the host Lipid metabolism is likely to be critical to the Wolbachia-host relationship. Both Wolbachia and insects lack cholesterol biosynthesis genes, and the Wolbachia genome also lacks fatty acid synthesis genes, so there is likely to be resource competition between Wolbachia and the host [49]. Our study showed that several genes involved in lipid synthesis were signi cantly upregulated in DmelW. ...
Wolbachia is a genus of intracellular symbiotic bacteria that are widely distributed in arthropods and nematodes. These maternally inherited bacteria regulate host reproductive systems in various ways to facilitate their vertical transmission. Since the identification of Wolbachia in many insects, the relationship between Wolbachia and the host has attracted great interest. Numerous studies have indicated that Wolbachia modifies a variety of biological processes in the host. Previous studies in Drosophila melanogaster ( D. mel ) have demonstrated that Wolbachia can affect spermatid differentiation, chromosome deposition, and sperm activity in the early stages of spermatogenesis, leading to sperm dysfunction. Here, we explored the putative effect of Wolbachia in sperm maturation using transcriptomic approaches to compare gene expression in Wolbachia -infected and Wolbachia -free D. mel adult testes. Our findings show that Wolbachia affects many biological processes in D. mel adult testes, and most of the differentially expressed genes involved in carbohydrate metabolism, lysosomal degradation, proteolysis, lipid metabolism, and immune response were upregulated in the presence of Wolbachia . In contrast, some genes that are putatively associated with cutin and wax biosynthesis and peroxisome pathways were downregulated. We did not find any differentially expressed genes that are predicted to be related to spermatogenesis in the datasets. This work provides additional information for understanding the Wolbachia -host intracellular relationships.
... The Wolbachia genome lacks genes for cholesterol and fatty acid synthesis, which may lead to competition with the host for these resources (Wu et al., 2004) and, as it has been observed, an increase in lipid concentration in infected hosts (Li et al., 2020). Our research revealed significant induction of ACSS3 and Hmgs1 by Wolbachia in the female gonads of Cpp, potentially leading to alteration in lipid metabolism. ...
Distinct lineages of the grasshopper Chorthippus parallelus (Orthoptera: Acrididae) form well‐known hybrid zones (HZs) both in the Pyrenees and the Alps mountain ranges in South Europe. These HZs represent unique experimental systems to identify “key genes” that maintain genetic boundaries between emerging species. The Iberian endemism C. p. erythropus (Cpe) and the subspecies C. p. parallelus (Cpp), widely distributed throughout the rest of Europe, overlap and form the Pyrenean HZ. Both subspecies differ morphologically, as well as in behavioral, mitochondrial, nuclear, and chromosomal traits, and in the strains of the maternally transmitted bacterial endosymbiont Wolbachia infecting them. This results in either unidirectional and bidirectional cytoplasmic incompatibility between both grasshopper subspecies, pointing out that Wolbachia clearly affects gene expression in the infected individuals. Here we explore how Wolbachia may modify the expression of some major genes involved in relevant pathways in Cpp in the Pyrenean HZ. We have analyzed, through molecular biomarkers, the physiological responses in C. parallelus individuals infected by Wolbachia , with particular attention to the energy metabolism, the immune system response, and the reproduction. qPCR was used to evaluate the expression of selected genes in the gonads of infected and uninfected adults of both sexes, since this tissue constitutes the main target of Wolbachia infection. Transcriptional analyses also showed differential sex‐dependent responses in most of the analyzed biomarkers in infected and noninfected individuals. We identified for the first time new sensitive biomarkers that might be involved in the reproductive barrier induced by Wolbachia in the hybrid zone.
... Comparative sequence analyses of bacteriophage WO revealed the possibility of large-scale horizontal gene transfer between Wolbachia coinfections in the same host [60]. Molecular mechanism used by Wolbachia to manipulate its host in terms of cytoplasmic incompatibility, feminization, parthenogenesis, male killing etc. remain elusive and has been speculated due to genes on extrachromosomal factors such as plasmids or bacteriophages [61,62]. Out of seventeen identified super-groups of Wolbachia, named A-Q , WO phage has been reported to infect Wolbachia belonging to super-group A, B, F and G [63,64]. ...
Lac insects are a specialized group of phytosuccivorous insects (Coccoidea: Tachardiidae) that secret resin of industrial importance having diverse applications. Due to unique biology, host preference and dispersal mechanisms, lac insects are expected to differentiate locally, forming geographic and host races without adequate morphological differentiation. 101 species of lac-insects and over 400 species of lac host plants have been reported but insects belonging to sub-family Tachardiinae are considered important for laksha-culture (lac insect farming). With a wide host-plant range and diverse habitat, the insects have developed a specialized ecosystem with multi-trophic complex of flora and fauna. Not only the lac insect but also the host plants and various biotic associations play a significant role in determining the quantity and quality of the produce. This insect being an obligate phloem sap sucker completes its life cycle on host plant species. Phloem sap is nutritionally unbalanced, as it is rich in carbohydrates but deficient in essential amino acids. Due to the scarcity of essential elements in phloem sap, endosymbionts are likely to co-evolve within the insect cell, while fulfilling their nutritional requirement. Implication of these intricate biotic associations on quantity and quality of the lac resin produced merits thorough understanding for sustained lac production.
... Comparative sequence analyses of bacteriophage WO revealed the possibility of large-scale horizontal gene transfer between Wolbachia coinfections in the same host [60]. Molecular mechanism used by Wolbachia to manipulate its host in terms of cytoplasmic incompatibility, feminization, parthenogenesis, male killing etc. remain elusive and has been speculated due to genes on extrachromosomal factors such as plasmids or bacteriophages [61,62]. Out of seventeen identified super-groups of Wolbachia, named A-Q , WO phage has been reported to infect Wolbachia belonging to super-group A, B, F and G [63,64]. ...
Lac insects are a specialized group of phytosuccivorous insects (Coccoidea: Tachardiidae) that secret resin of industrial importance having diverse applications. Due to unique biology, host preference and dispersal mechanisms, lac insects are expected to differentiate locally, forming geographic and host races without adequate morphological differentiation. 101 species of lac-insects and over 400 species of lac host plants have been reported but insects belonging to sub-family Tachardiinae are considered important for laksha-culture (lac insect farming). With a wide host-plant range and diverse habitat, the insects have developed a specialized ecosystem with multi-trophic complex of flora and fauna. Not only the lac insect but also the host plants and various biotic associations play a significant role in determining the quantity and quality of the produce. This insect being an obligate phloem sap sucker completes its life cycle on host plant species. Phloem sap is nutritionally unbalanced, as it is rich in carbohydrates but deficient in essential amino acids. Due to the scarcity of essential elements in phloem sap, endosymbionts are likely to co-evolve within the insect cell, while fulfilling their nutritional requirement. Implication of these intricate biotic associations on quantity and quality of the lac resin produced merits thorough understanding for sustained lac production.
... Meanwhile, the Wolbachia, an intracellular bacterium infecting nematodes, is among the most prevalent parasitic microorganisms affecting the reproductive parasite in the ecosystem (Taylor et al, 2018). Without Wolbachia colonization, some host species cannot propagate or even survive (Wu et al, 2004), as There was a strong mutualistic link between Wolbachia and nematode hosts' tissues (Engelstädter and Hurst, 2006). This co-operation helped them survive since Wolbachia is required for the nematode host's viability, growth and fertility, and in turn the nematode host provided the necessary amino acids for the Wolbachia's development (Foster et al, 2005). ...
... An important component of this analysis was the finding that this species did not have an orthologs of DNA ligase IV, even though the original annotation of the genome had suggested it did (Figure 1). Among the other interesting repair-related features we have found are: the presence of two MutL homologs in an intracellular bacteria Wolbachia pipientis wMel (40), the presence of two UvrA homologs in Deinococcus radiodurans (41) a n d Chlorobium tepidum (42), the absence of MutS and MutL from Mycobacterium tuberculosis (43), and the presence of multiple ligases for each chromosome in Agrobacterium tumefaciens (44). Continued surprises come from almost every genome. ...
Phylogenomics is a field in which genome analysis and evolutionary reconstructions are integrated. This integration is important because genome data is of great value in evolutionary reconstructions, because evolutionary analysis is critical for understanding and interpreting genomic data, and because there are feedback loops between evolutionary and genome analysis such that they need to be done in an integrated manner. In this paper I describe how I developed my particular phylogenomic approach under the guidance of my Ph.D advisor Philip C. Hanawalt. Since I was the first to use the term phylogenomics in a publication, I have decided to rename the field (at least temporarily) Philogenomics.
... Several pathways known to be involved in Wolbachia growth and metabolism were dysregulated in all Wolbachia-carrying lines, as expected. Genes involved in fatty acid synthesis and amino acid synthesis are absent from the genome of Wolbachia; therefore these processes are dysregulated when the bacterium is present [27][28][29][30][31] . The upregulation of proteasome proteins may be indicative of the need for a controlled breakdown of host proteins to increase amino acid availability 32 . ...
Some strains of the inherited bacterium Wolbachia have been shown to be effective at reducing the transmission of dengue virus (DENV) and other RNA viruses by Aedes aegypti in both laboratory and field settings and are being deployed for DENV control. The degree of virus inhibition varies between Wolbachia strains. Density and tissue tropism can contribute to these differences but there are also indications that this is not the only factor involved: for example, strains wAu and wAlbA are maintained at similar intracellular densities but only wAu produces strong DENV inhibition. We previously reported perturbations in lipid transport dynamics, including sequestration of cholesterol in lipid droplets, with strains wMel/wMelPop in Ae. aegypti. To further investigate the cellular basis underlying these differences, proteomic analysis of midguts was carried out on Ae. aegypti lines carrying strains wAu and wAlbA: with the hypothesis that differences in perturbations may underline Wolbachia-mediated antiviral activity. Surprisingly, wAu-carrying midguts not only showed distinct proteome perturbations when compared to non-Wolbachia carrying and wAlbA-carrying midguts but also wMel-carrying midguts. There are changes in RNA processing pathways and upregulation of a specific set of RNA-binding proteins in the wAu-carrying line, including genes with known antiviral activity. Lipid transport and metabolism proteome changes also differ between strains, and we show that strain wAu does not produce the same cholesterol sequestration phenotype as wMel. Moreover, in contrast to wMel, wAu antiviral activity was not rescued by cyclodextrin treatment. Together these results suggest that wAu could show unique features in its inhibition of arboviruses compared to previously characterized Wolbachia strains.
... Overrepresentation of GO categories involved in DNA biosynthetic process, deoxyribonucleotide biosynthetic process, carbohydrate derivative catabolic process, ribonucleoside-diphosphate reductase, cardiolipin biosynthetic process, integral component of plasma membrane, and CDP-diacylglycerol-glycerol-3-phosphate is consistent with these previous observations. As a result, PB could manifest because Wolbachia is dependent on the mutualistic relationship with its host to acquire all nutrients (66,67) and competition for resources could be detrimental to arbovirus infection. ...
A promising candidate for arbovirus control and prevention relies on replacing arbovirus-susceptible Aedes aegypti populations with mosquitoes that have been colonized by the intracellular bacterium Wolbachia and thus have a reduced capacity to transmit arboviruses. This reduced capacity to transmit arboviruses is mediated through a phenomenon referred to as pathogen blocking. Pathogen blocking has primarily been proposed as a tool to control dengue virus (DENV) transmission, however it works against a range of viruses, including Zika virus (ZIKV). Despite years of research, the molecular mechanisms underlying pathogen blocking still need to be better understood. Here, we used RNA-seq to characterize mosquito gene transcription dynamics in Ae. aegypti infected with the w Mel strain of Wolbachia that are being released by the World Mosquito Program in Medellín, Colombia. Comparative analyses using ZIKV-infected, uninfected tissues, and mosquitoes without Wolbachia revealed that the influence of w Mel on mosquito gene transcription is multifactorial. Importantly, because Wolbachia limits, but does not completely prevent, replication of ZIKV and other viruses in coinfected mosquitoes, there is a possibility that these viruses could evolve resistance to pathogen blocking. Therefore, to understand the influence of Wolbachia on within-host ZIKV evolution, we characterized the genetic diversity of molecularly barcoded ZIKV virus populations replicating in Wolbachia -infected mosquitoes and found that within-host ZIKV evolution was subject to weak purifying selection and, unexpectedly, loose anatomical bottlenecks in the presence and absence of Wolbachia . Together, these findings suggest that there is no clear transcriptional profile associated with Wolbachia -mediated ZIKV restriction, and that there is no evidence for ZIKV escape from this restriction in our system.
Author Summary
When Wolbachia bacteria infect Aedes aegypti mosquitoes, they dramatically reduce the mosquitoes’ susceptibility to infection with a range of arthropod-borne viruses, including Zika virus (ZIKV). Although this pathogen-blocking effect has been widely recognized, its mechanisms remain unclear. Furthermore, because Wolbachia limits, but does not completely prevent, replication of ZIKV and other viruses in coinfected mosquitoes, there is a possibility that these viruses could evolve resistance to Wolbachia -mediated blocking. Here, we use host transcriptomics and viral genome sequencing to examine the mechanisms of ZIKV pathogen blocking by Wolbachia and viral evolutionary dynamics in Ae. aegypti mosquitoes. We find complex transcriptome patterns that do not suggest a single clear mechanism for pathogen blocking. We also find no evidence that Wolbachia exerts detectable selective pressures on ZIKV in coinfected mosquitoes. Together our data suggest that it may be difficult for ZIKV to evolve Wolbachia resistance, perhaps due to the complexity of the pathogen blockade mechanism.
... We, therefore, sequenced the whole genome of wCcep, identifying two cifA and two cifB genes. In contrast, the genome of wMel contained only one cifA and one cifB (Wu et al., 2004). Crossing experiments showed that both wCcep and wMel are CI-inducing Wolbachia strains, and the strength of CI induced by wCcep (WT Â wC-ST) was not significantly different from that by wMel (WT Â wM-ST; Table 1). ...
Wolbachia can modulate the reproductive development of their hosts in multiple modes, and cytoplasmic incompatibility (CI) is the most well‐studied phenotype. The whitefly Bemisia tabaci is highly receptive to different Wolbachia strains: wCcep strain from the rice moth Corcyra cephalonica and wMel strain from the fruit fly Drosophila melanogaster could successfully establish and induce CI in transinfected whiteflies. Nevertheless, it is unknown what will happen when these two exogenous Wolbachia strains are co‐transinfected into a new host. Here, we artificially transinferred wCcep and wMel into the whitefly and established double‐ and singly‐transinfected B. tabaci isofemale lines. Reciprocal crossing experiments showed that wCcep and wMel induced a complex of CI phenotypes in the recipient host, including unidirectional and bidirectional CI. We next sequenced the whole genome of wCcep and performed a comparative analysis of the CI factor genes between wCcep and wMel, indicating that their cif genes were phylogenetically and structurally divergent, which can explain the crossing results. The amino acid sequence identity and structural features of Cif proteins may be useful parameters for predicting their function. Structural comparisons between CifA and CifB provide valuable clues for explaining the induction or rescue of CI observed in crossing experiments between transinfected hosts.
... Drosophila melanogaster (Wu et al., 2004) wAu NZ_LK055284.1 ...
... Par un mécanisme encore inconnu, la bactérie altère la différenciation sexuelle des juvéniles génétiquement mâles qui se différencient alors en femelles phénotypiques fonctionnelles, expliquant la thélygénie. Suite au séquençage de plusieurs souches de Wolbachia (Wu et al., 2004;Fenn and Blaxter, 2006), dont celles infectant plusieurs Isopodes terrestres (P. Grève, communication personnelle), il a été suggéré que des facteurs de virulence pourraient être libérés par la bactérie grâce à des systèmes de sécrétion de type I ou de type IV, vraisemblablement fonctionnels chez cette bactérie. ...
La différenciation sexuelle des Isopodes dépend d'une hormone sexuelle protéique, l'hormone androgène (HA), caractéristique des Malacostracés. Cet Insulin-Like Peptide suffit à induire par sa présence la différenciation mâle de ces Crustacés. Nous avons identifié in silico le transporteur circulant de l'HA, l'IGFBP-rP1, chez de nombreuses espèces d'Isopodes ainsi qu'à l'échelle des Crustacés. De la même façon, nous avons identifié deux récepteurs transmembranaires, l'IR1 et l'IR2, issus d'une duplication de gène spécifique des Malacostracés. Les patrons d'expression de ces gènes ont été étudiés sur notre espèce modèle, Armadillidium vulgare. Av-IGFBP-rP1 et Av-IR1 sont exprimés de manière ubiquiste et tout au long du développement. Av-IR2 est aussi exprimé à chaque stade de la différenciation mais ce transcrit est quasi-spécifique des glandes androgènes et ovaires. Une approche par ARNi a confirmé l'implication de ces trois protéines dans la voie de signalisation de l'HA. En effet, l'inhibition de l'HA, Av-IGFBP-rP1 et Av-IR1 provoquent l'hypertrophie des glandes androgènes, suggérant leur implication dans une boucle de rétro-contrôle de l'HA. L'inhibition de Av-IR2 semble seulement provoquer la différenciation d'ouvertures génitales femelles. Ces phénotypes sont comparables à ceux des intersexués mâles induits par la bactérie féminisante endogène Wolbachia. Nous montrons cependant que la bactérie altère seulement l'expression de l'HA et pas celle des récepteurs. Enfin, nous avons testé l'effet du bisphénol A mais nous n'observons pas d'altération de la différenciation sexuelle des larves lors d'expositions à ce perturbateur endocrinien exogène.
... The higher amino acid transport and metabolism in COG group indicate the role of the associated microbe in the nitrogen recycling process of the host organism (Schramm et al. 2003;Kjeldsen et al. 2012). Large numbers of mobile elements have been reported in the genomes of symbiotic bacteria (Wu et al. 2004;Haber et al. 2021), having a significant role in the evolution of bacterial genomes for symbiotic relationships with their hosts (Moran and Plague 2004). ...
Ascidians are a highly evolved invertebrate group enjoying a wide global distribution and are well known for their symbiotic association with microbial communities. Here, we analysed the genomic features of an ascidian associated bacterium, Bacillus aryabhattai MCCB 387 and compared its genomic features with the other three free-living B. aryabhattai reported from different ecological niches to underpin the gene repertoires involved in successful colonisation and symbiosis. In general, many ‘symbiosis factors’ genes have been observed in all four isolates demonstrating their potential to live with eukaryotic hosts. However, ascidian-associated B. aryabhattai MCCB 387 showed certain additional and unique features and metabolic specificity, which might have favoured and enhanced the ecological fitness of a host association. On detailed analysis, the gene for fibronectin proteins which helps to bind with integrins and facilitate cell–cell contact and possible colonisation is seen in ascidian-associated B. aryabhattai MCCB 387. The presence of one complete set of CRISPR-Cas region, Type I restriction-modification system and Toxin-antitoxin systems in MCCB 387 shows the genomic features favouring the symbiotic mode of life. MCCB 387 genome revealed a comparatively high number of Biosynthetic Gene Clusters (smBGCs) supporting the strain's secondary metabolite production potential, which might enhance fitness toward host-symbiont interaction. The genome of B. aryabhattai MCCB 387 is enriched with Mobile Genetic Elements (MGEs), suggestive of increased horizontal gene transfer between the host-microbial communities. Enriched MGEs might contribute to acquiring new genetic features and genome plasticity for the symbiont. Taken together, these genomic features might confer B. aryabhattai MCCB 387 an edge over other potential competitors, which likely enhances its ecological fitness and host colonisation.
... This cycling could be explained by natural selection, interactions with environment, or PCR artifacts. One could imagine scenarios where factors that limit Wolbachia densitiessuch as insect immunity (Kambris et al. 2009;Ye et al. 2013), access to nutritional resources (Geoghegan et al. 2017;Kabouridis et al. 2000;Wu et al. 2004), or access to cellular niches , prevent Wolbachia loads from rising too high despite selection on the symbiont to maximize transmission. This could also be the case if the rising relative densities might be associated with fitness costs in the vector, as shown previously (Ant et al. 2018). ...
Wolbachia is an insect endosymbiont being used for biological control in the mosquito Aedes aegypti because it causes cytoplasmic incompatibility (CI) and limits viral replication of dengue, chikungunya, and Zika viruses. While the genetic mechanism of pathogen blocking (PB) is not fully understood, the strength of both CI and PB are positively correlated with Wolbachia densities in the host. Wolbachia densities are determined by a combination of Wolbachia strain and insect genotype, as well as interactions with the environment. We employed both artificial selection and inbreeding with the goal of creating lines of Ae. aegypti with heritable and distinct Wolbachia densities so that we might better dissect the mechanism underlying PB. We were unable to shift the mean relative Wolbachia density in Ae. aegypti lines by either strategy, with relative densities instead tending to cycle over a narrow range. In lieu of this, we used Wolbachia densities in mosquito legs as predictors of relative densities in the remaining individual’s carcass. Because we worked with outbred mosquitoes, our findings indicate either a lack of genetic variation in the mosquito for controlling relative density, natural selection against extreme densities, or a predominance of environmental factors affecting densities. Our study reveals that there are moderating forces acting on relative Wolbachia densities that may help to stabilize density phenotypes post field release. We also show a means to accurately bin vector carcasses into high and low categories for non-DNA omics-based studies of Wolbachia-mediated traits.
... Genomes used are listed in Supplementary Table S6 (6,37,38,(59)(60)(61). We used Prokka v. 1.11 to annotate genomes, then extracted all single-copy genes that were the same length in each genome for phylogenetic analyses. ...
Divergent hosts often associate with intracellular microbes that influence their fitness. Maternally transmitted Wolbachia bacteria are the most common of these endosymbionts due largely to cytoplasmic incompatibility (CI) that kills uninfected embryos fertilized by Wolbachia-infected males. Closely related infections in females rescue CI, providing a relative fitness advantage that drives Wolbachia to high frequencies. One prophage-associated gene (cifA) governs rescue and two contribute to CI (cifA and cifB), but CI strength ranges from very strong to very weak for unknown reasons. Here, we investigate CI-strength variation and its mechanistic underpinnings in a phylogenetic context across 20 million years (MY) of Wolbachia evolution in Drosophila hosts diverged up to 50 MY. These Wolbachia encode diverse Cif proteins (100–7.4% pairwise similarity), and AlphaFold structural analyses suggest that CifB sequence similarities do not predict structural similarities. We demonstrate that cifB-transcript levels in testes explain CI strength across all but two focal systems. Despite phylogenetic discordance among cifs and the bulk of the Wolbachia genome, closely related Wolbachia tend to cause similar CI strengths and transcribe cifB at similar levels. This indicates that other non-cif regions of the Wolbachia genome modulate cif-transcript levels. CI strength also increases with the length of the host's larval life stage, presumably due to prolonged cif action. Our findings reveal that cifB-transcript levels largely explain CI strength while highlighting other covariates. Elucidating CI's mechanism contributes to our understanding of Wolbachia spread in natural systems and the efficacy of CI-based biocontrol of arboviruses and agricultural pests globally.
... One somatic tissue with a very high Wolbachia relative density in Ae. aegypti is the Malpighian Tubules [40]. These tissues are the main site of nitrogen secretion and as such they may especially facilitate Wolbachia growth, given that the symbiont primarily consumes host nucleotides [56], a large source of nitrogen. This same study also revealed pockets of cells within particular tissues, such as the midgut epithelia, thoracic ganglia and the salivary glands, that exhibit higher relative densities than the surrounding tissues [40]. ...
Background
The insect endosymbiotic bacterium Wolbachia is being deployed in field populations of the mosquito Aedes aegypti for biological control. This microbe prevents the replication of human disease-causing viruses inside the vector, including dengue, Zika and chikungunya. Relative Wolbachia densities may in part predict the strength of this ‘viral blocking’ effect. Additionally, Wolbachia densities may affect the strength of the reproductive manipulations it induces, including cytoplasmic incompatibility (CI), maternal inheritance rates or induced fitness effects in the insect host. High rates of CI and maternal inheritance and low rates of fitness effects are also key to the successful spreading of Wolbachia through vector populations and its successful use in biocontrol. The factors that control Wolbachia densities are not completely understood.
Methods
We used quantitative PCR-based methods to estimate relative density of the Wolbachia w AlbB strain in both the somatic and reproductive tissues of adult male and female mosquitoes, as well as in eggs. Using correlation analyses, we assessed whether densities in one tissue predict those in others within the same individual, but also across generations.
Results
We found little relationship among the relative Wolbachia densities of different tissues in the same host. The results also show that there was very little relationship between Wolbachia densities in parents and those in offspring, both in the same and different tissues. The one exception was with ovary–egg relationships, where there was a strong positive association. Relative Wolbachia densities in reproductive tissues were always greater than those in the somatic tissues. Additionally, the densities were consistent in females over their lifetime regardless of tissue, whereas they were generally higher and more variable in males, particularly in the testes.
Conclusions
Our results indicate that either stochastic processes or local tissue-based physiologies are more likely factors dictating Wolbachia densities in Ae. aegypti individuals, rather than shared embryonic environments or heritable genetic effects of the mosquito genome. These findings have implications for understanding how relative Wolbachia densities may evolve and/or be maintained over the long term in Ae. aegypti .
Graphical Abstract
... The genome of the original wMel transinfection (Walker et al., 2011) was very similar to both the wMel reference genome (Wu et al., 2004) and the wMel genome reported by Chrostek et al. (2013), differing from each by five and four polymorphic loci, respectively, consistent with the three strains having originated from same Drosophila stock (yw 67C23 ) less than 5000 generations ago (Supporting Information Table S2). Phylogenetic analysis placed these genomes in a single monophyletic cluster together with the other members of wMel clade III (Fig. 1). ...
Mosquito‐borne diseases remain a major cause of morbidity and mortality. Population replacement strategies involving the wMel strain of Wolbachia are being used widely to control mosquito‐borne diseases. However, these strategies may be influenced by temperature because wMel is vulnerable to heat. wMel infections in Drosophila melanogaster are genetically diverse, but few transinfections of wMel variants have been generated in Aedes aegypti. Here, we successfully transferred a wMel variant (termed wMelM) originating from a field‐collected D. melanogaster into Ae. aegypti. The new wMelM variant (clade I) is genetically distinct from the original wMel transinfection (clade III), and there are no genomic differences between wMelM in its original and transinfected host. We compared wMelM with wMel in its effects on host fitness, temperature tolerance, Wolbachia density, vector competence, cytoplasmic incompatibility and maternal transmission under heat stress in a controlled background. wMelM showed a higher heat tolerance than wMel, likely due to higher overall densities within the mosquito. Both wMel variants had minimal host fitness costs, complete cytoplasmic incompatibility and maternal transmission, and dengue virus blocking under laboratory conditions. Our results highlight phenotypic differences between Wolbachia variants and wMelM shows potential as an alternative strain in areas with strong seasonal temperature fluctuations.
... This competition for intracellular resources can impact both virus proliferation and potentially Wolbachia density (Caragata et al., 2013;Caragata et al., 2014). Wolbachia bacteria require fatty acids and cholesterol for their cellular metabolism and lack the biosynthetic capability to synthesize cholesterol, which suggests a need to obtain it from the host insect cell (Wu et al., 2004). The role of lipid transport in Wolbachia's capacity to inhibit DENV in vivo and in vitro has been previously demonstrated (Caragata et al., 2013;Koh et al., 2020). ...
Culicoides midges are hematophagous insects that transmit arboviruses of veterinary importance. These viruses include bluetongue virus (BTV) and epizootic hemorrhagic fever virus (EHDV). The endosymbiont Wolbachia pipientis Hertig spreads rapidly through insect host populations and has been demonstrated to inhibit viral pathogen transmission in multiple mosquito vectors. Here, we have demonstrated a replication inhibitory effect on BTV and EHDV in a Wolbachia (wAlbB strain)‐infected Culicoides sonorensis Wirth and Jones W8 cell line. Viral replication was significantly reduced by day 5 for BTV and by day 2 for EHDV as detected by real‐time polymerase chain reaction (RT‐qPCR) of the non‐structural NS3 gene of both viruses. Evaluation of innate cellular immune responses as a cause of the inhibitory effect showed responses associated with BTV but not with EHDV infection. Wolbachia density also did not play a role in the observed pathogen inhibitory effects, and an alternative hypothesis is suggested. Applications of Wolbachia‐mediated pathogen interference to impact disease transmission by Culicoides midges are discussed. Wolbachia infections in Culicoides sonorensis cells significantly reduced BTV and EHDV replication rates. Wolbachia virus inhibitory effects were associated with a host cell immune response but were not dependent upon intracellular Wolbachia density. The observed virus inhibitory effects suggest the potential to develop Wolbachia‐based disease control approaches for Culicoides vectored orbiviruses. Quantitative orbiviral replication as detected by RT‐qPCR inCulicoides cells with and without Wolbachiainfection. CulicoidesW8 cell line with Wolbachia infection (W8‐w; blue) and without (W8; red) from 0 to 7 days post infection (DPI) with BTV and EHDV. Bars represent means ± standard error (SEM) calculated across two independent replicate experiments. (*p < 0.05, ** p < 0.01).
... 52,53 Indirect competition may exist among these bacteria, forming a Wolbachia-host-microbe interrelationship. During this process, Wolbachia may secrete some compounds through secretion systems that pass through the cell to affect other bacteria, 54 and may also inhibit the density of some bacteria, resulting in their loss during transmission. 55 Second, Wolbachia may stimulate the host's immune response (such as producing antimicrobial peptides and other compounds) to regulate the bacterial community. ...
BACKGROUND
Wolbachia has been developed as an effective tool to suppress insect pests and arbovirus transmission. Recently, the brown planthopper Nilaparvata lugens, a serious agricultural pest, has been successfully transinfected with Wolbachia wStri strain from Laodelphax striatellus. However, before conducting the field experiments, the impacts of wStri on the bacterial microbiota in N. lugens and how it differs from native Wolbachia wLug strain have not been clarified.
RESULTS
Here, we found that wStri reduced bacterial diversity and shaped bacterial community structure more than wLug in both developmental stage and different adult tissues. Overall, the relative abundance of Wolbachia was negatively correlated with bacterial diversity, but the bacterial diversity gradually decreased only when the relative abundance of Wolbachia was higher than 60%. Further analysis found that wStri reduced species richness of other bacteria but not their evenness. wStri infection also affected many bacterial functions (e.g., amino acid metabolism & signaling and cellular processes) in the developmental stages, with a stronger effect than wLug in nymphs. Moreover, although Wolbachia occupied a high relative abundance in infected individuals, Acinetobacter was consistently a core part of microbiome.
CONCLUSION
These results showed the significant impacts of recently introduced wStri on bacterial microbiota in N. lugens, with the effects differing from native wLug. This study will aid in understanding the relationship between Wolbachia, its host and the host’s microbiota, and provide a reference for future field experiments.
... Lipid droplets (LDs) containing a few exclusive structural proteins (Perilipin 1, 2, and 3) and a fatty acid monolayer are exclusively found to present in a variety of organisms including DENV. These have been found to provide immunological defense of Aedes mosquitoes [53][54][55]. ...
Dengue remains one of the most serious and widespread mosquito-borne viral infections in human beings, with serious health problems or even death. About 50 to 100 million people are newly infected annually, with almost 2.5 billion people living at risk and resulting in 20,000 deaths. Dengue virus infection is especially transmitted through bites of Aedes mosquitos, hugely spread in tropical and subtropical environments, mostly found in urban and semiurban areas. Unfortunately, there is no particular therapeutic approach, but prevention, adequate consciousness, detection at earlier stage of viral infection, and appropriate medical care can lower the fatality rates. This review offers a comprehensive view of production, transmission, pathogenesis, and control measures of the dengue virus and its vectors.
... Bacteria of the genus Wolbachia are a powerful model for host-microbe interaction studies 1 . Genomic analysis suggests that Wolbachia lacks a series of essential enzymes, including part of the glycolytic pathway, which indicates that the bacterium relies on its host cell machinery for energy production and other metabolic functions [2][3][4][5] . The obligate symbiosis between insects and bacteria are among the most complex associations; host and symbiont are integrated into a tidy biological entity and are unable to survive otherwise 6,7 . ...
In the present work, we established two novel embryonic cell lines from the mosquito Aedes fluviatilis containing or not the naturally occurring symbiont bacteria Wolbachia, which were called w Aflu1 and Aflu2, respectively. We also obtained wAflu1 without Wolbachia after tetracycline treatment, named wAflu1.tet. Morphofunctional characterization was performed to help elucidate the symbiont-host interaction in the context of energy metabolism regulation and molecular mechanisms of the immune responses involved. The presence of Wolbachia pipientis improves energy performance in A. fluviatilis cells; it affects the regulation of key energy sources such as lipids, proteins, and carbohydrates, increasing the speed of cell proliferation, making the distribution of actin more peripheral and with more extensions that come into contact with neighboring cells. Additionally, innate immunity mechanisms were activated, showing that the w Aflu1 and wAflu1.tet cells are responsive after the stimulus using Gran negative bacteria. Therefore, this work confirms the natural, mutually co-regulating symbiotic relationship between W. pipientis and A. fluviatilis , modulating the host metabolism and immune pathway activation. The results presented here add important resources to the current knowledge of Wolbachia-arthropod interactions.
... All Wolbachia reads were identified and isolated from the raw read data of the I. elegans genome project [56]. The wEle1 assembly was built by mapping reads to two previously sequenced Wolbachia genomes (wPip [57] and wMel [58]) using bwa mem version 0.7.8 [59]. The properly mapped pairs were extracted using samtools 1.8. ...
Background
Evolutionary processes can cause strong spatial genetic signatures, such as local loss of genetic diversity, or conflicting histories from mitochondrial versus nuclear markers. Investigating these genetic patterns is important, as they may reveal obscured processes and players. The maternally inherited bacterium Wolbachia is among the most widespread symbionts in insects. Wolbachia typically spreads within host species by conferring direct fitness benefits, and/or by manipulating its host reproduction to favour infected over uninfected females. Under sufficient selective advantage, the mitochondrial haplotype associated with the favoured maternally-inherited symbiotic strains will spread (i.e. hitchhike), resulting in low mitochondrial genetic variation across the host species range.
Method
The common bluetail damselfly ( Ischnura elegans: van der Linden, 1820) has recently emerged as a model organism for genetics and genomic signatures of range expansion during climate change. Although there is accumulating data on the consequences of such expansion on the genetics of I. elegans , no study has screened for Wolbachia in the damselfly genus Ischnura . Here, we present the biogeographic variation in Wolbachia prevalence and penetrance across Europe and Japan (including samples from 17 populations), and from close relatives in the Mediterranean area (i.e. I. genei : Rambur, 1842; and I. saharensis : Aguesse, 1958).
Results
Our data reveal (a) multiple Wolbachia -strains, (b) potential transfer of the symbiont through hybridization, (c) higher infection rates at higher latitudes, and (d) reduced mitochondrial diversity in the north-west populations, indicative of hitchhiking associated with the selective sweep of the most common strain. We found low mitochondrial haplotype diversity in the Wolbachia -infected north-western European populations (Sweden, Scotland, the Netherlands, Belgium, France and Italy) of I. elegans , and, conversely, higher mitochondrial diversity in populations with low penetrance of Wolbachia (Ukraine, Greece, Montenegro and Cyprus). The timing of the selective sweep associated with infected lineages was estimated between 20,000 and 44,000 years before present, which is consistent with the end of the last glacial period about 20,000 years.
Conclusions
Our findings provide an example of how endosymbiont infections can shape spatial variation in their host evolutionary genetics during postglacial expansion. These results also challenge population genetic studies that do not consider the prevalence of symbionts in many insects, which we show can impact geographic patterns of mitochondrial genetic diversity.
... Wolbachia has a small genome (0.8-1.7 Mbp) with large segments of mobile and repetitive DNA, which is uncommon in vertically transmitted (generally from mother to offspring) organisms [4,23]. Despite the erosive genomic processes due to host restriction and acquisition maintenance, these repetitive host DNA sequences are supposed to play a major role in the evolution of Wolbachia [24]. Balance among vertical transmission, host switching, recombination insertion sequences and bacteriophage sequences helps in the adaptation and global distribution of Wolbachia [4,22]. ...
Background
Wolbachia is an obligate intracellular maternally transmitted, gram-negative bacterium which forms a spectrum of endosymbiotic relationships from parasitism to obligatory mutualism in a wide range of arthropods and onchocercid nematodes, respectively. In arthropods Wolbachia produces reproductive manipulations such as male killing, feminization, parthenogenesis and cytoplasmic incompatibility for its propagation and provides an additional fitness benefit for the host to protect against pathogens, whilst in onchocercid nematodes, apart from the mutual metabolic dependence, this bacterium is involved in moulting, embryogenesis, growth and survival of the host.
Methods
This review details the molecular data of Wolbachia and its effect on host biology, immunity, ecology and evolution, reproduction, endosymbiont-based treatment and control strategies exploited for filariasis. Relevant peer-reviewed scientic papers available in various authenticated scientific data bases were considered while writing the review.
Conclusions
The information presented provides an overview on Wolbachia biology and its use in the control and/or treatment of vectors, onchocercid nematodes and viral diseases of medical and veterinary importance. This offers the development of new approaches for the control of a variety of vector-borne diseases.
Graphic Abstract
... The first sequenced Wolbachia genome, wMel strain in Drosophila melanogaster from supergroup A, revealed a small 1.3 megabase (Mb) genome littered with mobile genetic elements (Wu et al., 2004). The genome sequences from wBm of Brugia malayi (Foster et al., 2005), wPip of C. pipiens (Klasson et al., 2008), wRi of D. simulans (Klasson et al., 2009), and wAlbB of Aedes albopictus (Mavingui et al., 2012) followed thereafter. ...
The most widespread intracellular bacteria in the animal kingdom are maternally inherited endosymbionts of the genus Wolbachia. Their prevalence in arthropods and nematodes worldwide and stunning arsenal of parasitic and mutualistic adaptations make these bacteria a biological archetype for basic studies of symbiosis and applied outcomes for curbing human and agricultural diseases. Here, we conduct a summative, centennial analysis of living in the Wolbachia world. We synthesize literature on Wolbachia’s host range, phylogenetic diversity, genomics, cell biology, and applications to filarial, arboviral, and agricultural diseases. We also review the mobilome of Wolbachia including phage WO and its essentiality to hallmark reproductive phenotypes in arthropods. Finally, the Wolbachia system is an exemplar for discovery-based science education using biodiversity, biotechnology, and bioinformatics lessons. As we approach a century of Wolbachia research, the interdisciplinary science of this symbiosis stands as a model for consolidating and teaching the integrative rules of endosymbiotic life.
... All Wolbachia reads were identi ed and isolated from the raw read data of the I. elegans genome project [46]. The wEle1 assembly was built by mapping reads to two previously sequenced Wolbachia genomes (wPip [87] and wMel [88]) using bwa mem version 0.7.8 [89]. The properly mapped pairs were extracted using samtools 1.8. ...
Background Evolutionary processes can cause strong spatial genetic signatures, such as local loss of genetic diversity, or conflicting histories from mitochondrial versus nuclear markers. Investigating these genetic patterns is important, as they may reveal obscured processes and players. The maternally inherited bacterium Wolbachia is among the most widespread symbionts in insects. Wolbachia typically spreads within host species by conferring direct fitness benefits, or by manipulating its host reproduction to favour infected over uninfected females. Under sufficient selective advantage, the mitochondrial haplotype associated with the favoured symbiotic strains will spread (i.e. hitchhike), resulting in low mitochondrial genetic variation across the host species range. The common bluetail damselfly (Ischnura elegans: van der Linden, 1820) has recently emerged as a model organism of the genetics and genomic signatures of range expansion during climate change. Although there is accumulating data on the consequences of such expansion on the genetic of I. elegans, no study has screened for Wolbachia in the damselfly genus Ischnura. Here, we present the biogeographic variation in Wolbachia prevalence and penetrance in 17 I. elegans populations across Europe and Japan, and from close relatives in the Mediterranean area (i.e. I. genei: Rambur, 1842; and I. saharensis: Aguesse, 1958).
Results Our data reveal (a) multiple Wolbachia-strains, (b) potential transfer of the symbiont through hybridization, (c) higher infection rates at higher latitudes, and (d) reduced mitochondrial diversity in the north-west populations, indicative of hitchhiking associated with the selective sweep of the most common strain. We found low mitochondrial haplotype diversity in the Wolbachia-infected north-western European populations (Sweden, Scotland, the Netherlands, Belgium, France and Italy) of I. elegans, and, conversely, higher mitochondrial diversity in populations with low penetrance of Wolbachia (Ukraine, Greece, Montenegro and Cyprus). The timing of the selective sweep associated with infected lineages was estimated between 20 000 to 44 000 years before present, which is consistent with the end of the last glacial period about 20 000 ya.
Conclusions Our findings provide an example of how endosymbiont infections ca shape spatial variation in their host evolutionary genetics during postglacial expansion. These results also challenge population genetic studies that do not consider the prevalence of symbionts in many insects, which can impact geographic patterns of mitochondrial genetic diversity.
... Loss of DNA repair genes has been shown to co-occur with elevated mutation rates in intracellular bacteria [105][106][107], and may explain our observations. The mismatch repair protein-encoding loci mutS and mutL are universally lacking in Spiroplasma, but are present in the slower-evolving symbionts Wolbachia wMel and Buchnera aphidicola (Fig. 7) [108][109][110]. In Escherichia coli, hypermutator strains that have lost such loci have a ~10-300-fold increase of spontaneous mutation rates [111,112], which is comparable to the approximately two orders of magnitude difference between substitution rates of Buchnera and Spiroplasma (Fig. 2). ...
Spiroplasma is a genus of Mollicutes whose members include plant pathogens, insect pathogens and endosymbionts of animals. Spiroplasma phenotypes have been repeatedly observed to be spontaneously lost in Drosophila cultures, and several studies have documented a high genomic turnover in Spiroplasma symbionts and plant pathogens. These observations suggest that Spiroplasma evolves quickly in comparison to other insect symbionts. Here, we systematically assess evolutionary rates and patterns of Spiroplasma poulsonii , a natural symbiont of Drosophila . We analysed genomic evolution of s Hy within flies, and s Mel within in vitro culture over several years. We observed that S. poulsonii substitution rates are among the highest reported for any bacteria, and around two orders of magnitude higher compared with other inherited arthropod endosymbionts. The absence of mismatch repair loci mutS and mutL is conserved across Spiroplasma , and likely contributes to elevated substitution rates. Further, the closely related strains s Mel and s Hy (>99.5 % sequence identity in shared loci) show extensive structural genomic differences, which potentially indicates a higher degree of host adaptation in s Hy, a protective symbiont of Drosophila hydei . Finally, comparison across diverse Spiroplasma lineages confirms previous reports of dynamic evolution of toxins, and identifies loci similar to the male-killing toxin Spaid in several Spiroplasma lineages and other endosymbionts. Overall, our results highlight the peculiar nature of Spiroplasma genome evolution, which may explain unusual features of its evolutionary ecology.
Background
Members of the Acari order, commonly known as mites, play a significant role as agricultural pests. Among these, the Tetranychidae family stands out due to its remarkable diversity, surrounding approximately 1200 species capable of infesting over 4000 plant species. By feeding on plant tissues, these mites directly harm crops and can also serve as vectors for viral pathogens, posing a substantial threat to global food security. In this study, we investigated the unexplored virome of Tetranychus truncatus exploring how biotic factors (Spiroplasma and Wolbachia endosymbionts), and abiotic stresses (abamectin and temperature) affect virus dynamics.
Results
Our metatranscriptomics analyses revealed sequences related to important arthropod- and crop-infecting viral families, including the known plant-pathogenic Potato Y virus and Cherry A virus strains and fourteen new species. Notably, abamectin treatment correlated with the absence of Potato virus Y and TtDV-2 virus, suggesting this pesticide impacts viral diversity. Interestingly, single infections of Wolbachia or Spiroplasma significantly decreased both the diversity and the abundance of viruses, with the greatest effect on dicistroviruses, indicating for the first time the potential of Spiroplasma to restrict viral infections. Surprisingly, Wolbachia-Spiroplasma co-infection leads to the loss of the virus restriction effect. Wolbachia-exclusive and Spiroplasma-exclusive responsive genes showed enrichment for similar pathways, with piRNA and autophagy enriched in up-regulated genes. In contrast, lipid metabolic processes were enriched in down-regulated elements.
Conclusions
Overall, our study describes the T. truncatus virome, unveiling the considerable influence of its microbiome, including Wolbachia and Spiroplasma, and Abamectin pesticide on both virus diversity and abundance.
Intracellular bacteria use secreted effector proteins to modify host biology and facilitate infection. For many of these microbes, a particular eukaryotic domain—the ankyrin repeat (ANK)—plays a central role in specifying the host proteins and pathways targeted by the microbe. While we understand much of how some ANKs function in model organisms like Legionella and Coxiella, the understudied Rickettsiales species harbor many proteins with ANKs, some of which play critical roles during infection. This minireview is meant to organize and summarize the research progress made in understanding some of these Rickettsiales ANKs as well as document some of the techniques that have driven much of this progress.
The most common intracellular bacterial infection is Wolbachia pipientis, a microbe that manipulates host reproduction and is used in control of insect vectors. Phenotypes induced by Wolbachia have been studied for decades and range from sperm-egg incompatibility to male killing. How Wolbachia alters host biology is less well understood. Previously, we characterized the first Wolbachia effector–WalE1, which encodes an alpha-synuclein domain at the N terminus. Purified WalE1 sediments with and bundles actin and when heterologously expressed in flies, increases Wolbachia titer in the developing oocyte. In this work, we first identify the native expression of WalE1 by Wolbachia infecting both fly cells and whole animals. WalE1 appears as aggregates in the host cell cytosol. We next show that WalE1 co-immunoprecipitates with the host protein Past1, although might not directly interact with it, and that WalE1 manipulates host endocytosis. Yeast expressing WalE1 show deficiency in uptake of FM4-64 dye, and flies harboring mutations in Past1 or overexpressing WalE1 are sensitive to AgNO3, a hallmark of endocytosis defects. We also show that flies expressing WalE1 suffer from endocytosis defects in larval nephrocytes. Finally, we also show that Past1 null flies harbor more Wolbachia overall and in late egg chambers. Our results identify interactions between Wolbachia and a host protein involved in endocytosis and point to yet another important host cell process impinged upon by Wolbachia’s WalE1 effector.
Simple Summary
The usefulness of Wolbachia-based control methods for several insect orders is investigated in this synthesis, with a particular emphasis on sterile insect technique (SIT) and incompatible insect technique (IIT). Strong control tactics and financing are required, as demonstrated by the integration of SIT into the management of Drosophila suzukii and the evaluation of a new SIT/IIT combination against Aedes mosquitoes. The potential of Wolbachia includes biological vector control in agriculture, particularly defence against pests that affect rice. Examining host-shifting dynamics and phenotypic impacts, especially in scale insects, highlights the ecological connectivity that is essential to comprehending the complexity of Wolbachia. The summary discusses the worldwide distribution of Wolbachia, focusing on genetics, medicinal uses, and mutualistic and parasitic adaptations. With developments in gene functional assays and multiomics, Wolbachia research is changing and becoming a paradigm for microbial symbiosis, with profound effects on education and translational research.
Abstract
Effective in a variety of insect orders, including dipteran, lepidopteran, and hemipteran, Wolbachia-based control tactics are investigated, noting the importance of sterile and incompatible insect techniques. Encouraging approaches for controlling Aedes mosquitoes are necessary, as demonstrated by the evaluation of a new SIT/IIT combination and the incorporation of SIT into Drosophila suzukii management. For example, Wolbachia may protect plants from rice pests, demonstrating its potential for agricultural biological vector management. Maternal transmission and cytoplasmic incompatibility dynamics are explored, while Wolbachia phenotypic impacts on mosquito and rice pest management are examined. The importance of host evolutionary distance is emphasised in recent scale insect research that addresses host-shifting. Using greater information, a suggested method for comprehending Wolbachia host variations in various contexts emphasises ecological connectivity. Endosymbionts passed on maternally in nematodes and arthropods, Wolbachia are widely distributed around the world and have evolved both mutualistic and parasitic traits. Wolbachia is positioned as a paradigm for microbial symbiosis due to advancements in multiomics, gene functional assays, and its effect on human health. The challenges and opportunities facing Wolbachia research include scale issues, ecological implications, ethical conundrums, and the possibility of customising strains through genetic engineering. It is thought that cooperative efforts are required to include Wolbachia-based therapies into pest management techniques while ensuring responsible and sustainable ways.
Wolbachia pipientis had its scientific debut nearly a century ago and has recently emerged as a target for therapeutic treatment of filarial infections and an attractive tool for control of arthropod pests. Wolbachia was known as a biological entity before DNA was recognized as the molecule that carries the genetic information on which life depends, and before arthropods and nematodes were grouped in the Ecdysozoa. Today, some investigators consider Wolbachia the most abundant endosymbiont on earth, given the numbers of its hosts and its diverse mutualistic, commensal, and parasitic roles in their life histories. Recent advances in molecular technologies have revolutionized our understanding of Wolbachia and its associated reproductive phenotypes. New models have emerged for its investigation, and substantial progress has been made towards Wolbachia-based interventions in medicine and agriculture. Here I introduce Wolbachia, with a focus on aspects of its biology that are covered in greater detail in subsequent chapters.
The evolution of endosymbionts and their hosts can lead to highly dynamic interactions with varying fitness effects for both the endosymbiont and host species. Wolbachia, a ubiquitous endosymbiont of arthropods and nematodes, can have both beneficial and detrimental effects on host fitness. We documented the occurrence and patterns of transmission of Wolbachia within the Hawaiian Drosophilidae and examined the potential contributions of Wolbachia to the rapid diversification of their hosts. Screens for Wolbachia infections across a minimum of 140 species of Hawaiian Drosophila and Scaptomyza revealed species-level infections of 20.0%, and across all 399 samples, a general infection rate of 10.3%. Among the 44 Wolbachia strains we identified using a modified Wolbachia multi-locus strain typing scheme, 30 (68.18%) belonged to supergroup B, five (11.36%) belonged to supergroup A, and nine (20.45%) had alleles with conflicting supergroup assignments. Co-phylogenetic reconciliation analysis indicated that Wolbachia strain diversity within their endemic Hawaiian Drosophilidae hosts can be explained by vertical (e.g., co-speciation) and horizontal (e.g., host switch) modes of transmission. Results from stochastic character trait mapping suggest that horizontal transmission is associated with the preferred oviposition substrate of the host, but not the host’s plant family or island of occurrence. For Hawaiian Drosophilid species of conservation concern, with 13 species listed as endangered and 1 listed as threatened, knowledge of Wolbachia strain types, infection status, and potential for superinfection could assist with conservation breeding programs designed to bolster population sizes, especially when wild populations are supplemented with laboratory-reared, translocated individuals. Future research aimed at improving the understanding of the mechanisms of Wolbachia transmission in nature, their impact on the host, and their role in host species formation may shed light on the influence of Wolbachia as an evolutionary driver, especially in Hawaiian ecosystems.
Scrub typhus is the leading source of febrile illness in endemic countries due to infection with Orientia tsutsugamushi ( Ot ), a seriously understudied intracellular bacterium. Pulmonary complications in patients are common and can develop into life threatening conditions. The diverse antigenicity of Ot genotypes and inter-strain differences seem to be connected to varied virulence and clinical outcomes; however, detailed studies of strain-related pulmonary immune responses in human patients or experimental animals are lacking. In this study, we used two clinically prevalent bacterial strains, Karp and Gilliam, and revealed cellular immune responses in inflamed lungs and potential biomarkers of disease severity. We found that outbred CD-1 mice were highly susceptible to both Karp and Gilliam strains; however, C57BL/6 (B6) mice were susceptible to Karp, but resistant to Gilliam (with self-limiting infection), corresponding to their tissue bacterial burdens and lung pathological changes. Multicolor flow cytometric analyses of perfused B6 mouse lungs revealed robust and sustained influx and activation of innate immune cells (monocytes, macrophages, neutrophils, and NK cells), followed by those of CD4 ⁺ and CD8 ⁺ T cells, during Karp infection, but such responses were greatly attenuated during Gilliam infection. The robust cellular responses in Karp-infected B6 mice were positively correlated with significantly early and high levels of serum cytokine/chemokine protein levels (CXCL1, CCL2/3/5, and G-CSF), as well as pulmonary gene expression ( CXCL1/2, CCL2/3/4, and IFNγ ). In vitro infection of B6 mouse-derived primary macrophages also revealed bacterial strain-dependent immune gene expression profiles. This study provided the first lines of evidence that highlighted differential tissue cellular responses against Karp vs. Gilliam infection, offering a framework for future investigation of Ot strain-related mechanisms of disease pathogenesis vs. infection control.
Authors Summary
Orientia tsutsugamushi ( Ot ) infection-induced scrub typhus is a leading cause of febrile illness in endemic countries. Research on Ot strain-related disease outcomes or immune signatures in tissue and blood samples is very limited. Using two clinically prevalent strains (Karp and Gilliam), we examined host susceptibility in inbred and outbred mouse models and provided new evidence for the activation of pulmonary immune cell subsets during the acute stages of infection. While Gilliam-infected C57BL/6 (B6) mice developed self-limiting infection, mild cellular responses, and tissue injury, Karp infection led to a strong and sustained activation of innate immune cells, followed by extensive influx of activated T cells, which correlated to protein levels of inflammatory cytokines/chemokines in serum samples. We also provided in vitro evidence for Ot strain-dependent immune gene profiles, indicating differential macrophage responses to Karp versus Gilliam bacteria. This is the first comparison of different scrub typhus mouse models with in-depth analyses of cellular responses in inflamed lungs, offering novel insights into potential mechanisms of disease progression versus infection control related to Ot strains and laying the foundation for future investigations.
Endosymbioses influence host physiology, reproduction, and fitness, but these relationships require efficient microbe transmission between host generations to persist. Maternally transmitted Wolbachia are the most common known endosymbionts,1 but their frequencies vary widely within and among host populations for unknown reasons.2,3 Here, we integrate genomic, cellular, and phenotypic analyses with mathematical models to provide an unexpectedly simple explanation for global wMel Wolbachia prevalence in Drosophila melanogaster. Cooling temperatures decrease wMel cellular abundance at a key stage of host oogenesis, producing temperature-dependent variation in maternal transmission that plausibly explains latitudinal clines of wMel frequencies on multiple continents. wMel sampled from a temperate climate targets the germline more efficiently in the cold than a recently differentiated tropical variant (∼2,200 years ago), indicative of rapid wMel adaptation to climate. Genomic analyses identify a very narrow list of wMel alleles-most notably, a derived stop codon in the major Wolbachia surface protein WspB-that underlie thermal sensitivity of cellular Wolbachia abundance and covary with temperature globally. Decoupling temperate wMel and host genomes further reduces transmission in the cold, a pattern that is characteristic of host-microbe co-adaptation to a temperate climate. Complex interactions among Wolbachia, hosts, and the environment (GxGxE) mediate wMel cellular abundance and maternal transmission, implicating temperature as a key determinant of Wolbachia spread and equilibrium frequencies, in conjunction with Wolbachia effects on host fitness and reproduction.4,5 Our results motivate the strategic use of locally selected wMel variants for Wolbachia-based biocontrol efforts, which protect millions of individuals from arboviruses that cause human disease.6.
In the present work, we established two novel embryonic cell lines from the mosquito Aedes fluviatilis containing or not the naturally occurring symbiont bacteria Wolbachia, which were called wAflu1 and Aflu2, respectively. We also obtained wAflu1 without Wolbachia after tetracycline treatment, named wAflu1.tet. Morphofunctional characterization was performed to help elucidate the symbiont-host interaction in the context of energy metabolism regulation and molecular mechanisms of the immune responses involved. The presence of Wolbachia pipientis improves energy performance in A. fluviatilis cells; it affects the regulation of key energy sources such as lipids, proteins, and carbohydrates, making the distribution of actin more peripheral and with extensions that come into contact with neighboring cells. Additionally, innate immunity mechanisms were activated, showing that the wAflu1 and wAflu1.tet cells are responsive after the stimulus using Gram negative bacteria. Therefore, this work confirms the natural, mutually co-regulating symbiotic relationship between W. pipientis and A. fluviatilis, modulating the host metabolism and immune pathway activation. The results presented here add important resources to the current knowledge of Wolbachia-arthropod interactions.
Determining the phylogenetic origin of mitochondria is key to understanding the ancestral mitochondrial symbiosis and its role in eukaryogenesis. However, the precise evolutionary relationship between mitochondria and their closest bacterial relatives remains hotly debated. The reasons include pervasive phylogenetic artefacts, as well as limited protein and taxon sampling. Here, we developed a new model of protein evolution that accommodates both across-site and across-branch compositional heterogeneity. We applied this site-and-branch-heterogeneous model (MAM60 + GFmix) to a considerably expanded dataset that comprises 108 mitochondrial proteins of alphaproteobacterial origin, and novel metagenome-assembled genomes from microbial mats, microbialites, and sediments. The MAM60 + GFmix model fits the data much better and agrees with analyses of compositionally homogenized datasets with conventional site-heterogenous models. The consilience of evidence thus suggests that mitochondria is sister to the Alphaproteobacteria to the exclusion of MarineProteo1 and Magnetococcia . We also show that the ancestral presence of a crista-developing MICOS complex (a Mitofilin domain-containing Mic60) supports this relationship.
Anaplasma marginale is a vector-borne pathogen that causes a disease known as anaplasmosis. No sequenced genomes of Brazilian strains are yet available. The aim of this work was to compare whole genomes of Brazilian strains of A. marginale (Palmeira and Jaboticabal) with genomes of strains from other regions (USA and Australia strains). Genome sequencing of Brazilian strains was performed by means of next-generation sequencing. Reads were mapped using the genome of the Florida strain of A. marginale as a reference sequence. Single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs) were identified. The data showed that two Brazilian strains grouped together in one particular clade, which grouped in a larger American group together with North American strains. Moreover, some important differences in surface proteins between the two Brazilian isolates can be discerned. These results shed light on the evolutionary history of A. marginale and provide the first genome information on South American isolates. Assessing the genome sequences of strains from different regions is essential for increasing knowledge of the pan-genome of this bacteria.
Animals serve as hosts for complex communities of microorganisms, including endosymbionts that live inside their cells. Wolbachia bacteria are perhaps the most common endosymbionts, manipulating host reproduction to propagate. Many Wolbachia cause intense cytoplasmic incompatibility (CI) that promotes their spread to high and relatively stable frequencies. Wolbachia that cause weak or no CI tend to persist at intermediate, often variable, frequencies. Wolbachia could also contribute to host reproductive isolation (RI), although current support for such contributions is limited to a few systems. To test for Wolbachia frequency variation and effects on host RI, we sampled several local Prosapia ignipectus (Fitch)(Hemiptera: Cercopidae) spittlebug populations in the northeastern USA over two years, including closely juxtaposed Maine populations with different monomorphic color forms, “black” and “lined”. We discovered a group-B Wolbachia (wPig) infecting P. ignipectus that diverged from group-A Wolbachia, like model wMel and wRi strains in Drosophila, 6 to 46 MYA. Populations of the sister species Prosapia bicincta (Say) from Hawaii and Florida are uninfected, suggesting that P. ignipectus acquired wPig after their initial divergence. wPig frequencies were generally high and variable among sites and between years. While phenotyping wPig effects on host reproduction is not currently feasible, the wPig genome contains three divergent sets of CI loci, consistent with high wPig frequencies. Finally, Maine monomorphic black and monomorphic lined populations of P. ignipectus share both wPig and mtDNA haplotypes, implying no apparent effect of wPig on the maintenance of this morphological contact zone. We hypothesize P. ignipectus acquired wPig horizontally as observed for many Drosophila species, and that significant CI and variable transmission produce high but variable wPig frequencies.
The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene ( wsp ) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli . Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.
The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution.
Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human
genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
Determining protein functions from genomic sequences is a central goal of bioinformatics. We present a method based on the assumption that proteins that function together in a pathway or structural complex are likely to evolve in a correlated fashion. During evolution, all such functionally linked proteins tend to be either preserved or eliminated in a new species. We describe this property of correlated evolution by characterizing each protein by its phylogenetic profile, a string that encodes the presence or absence of a protein in every known genome. We show that proteins having matching or similar profiles strongly tend to be functionally linked. This method of phylogenetic profiling allows us to predict the function of uncharacterized proteins.
We describe here the complete genome sequence (1,111,523 base pairs) of
the obligate intracellular parasite Rickettsia prowazekii, the causative
agent of epidemic typhus. This genome contains 834 protein-coding genes. The
functional profiles of these genes show similarities to those of mitochondrial
genes: no genes required for anaerobic glycolysis are found in either
R. prowazekii or mitochondrial genomes, but a complete set of genes encoding
components of the tricarboxylic acid cycle and the respiratory-chain complex
is found in R. prowazekii. In effect, ATP production in Rickettsia
is the same as that in mitochondria. Many genes involved in the biosynthesis
and regulation of biosynthesis of amino acids and nucleosides in free-living
bacteria are absent from R. prowazekii and mitochondria. Such genes
seem to have been replaced by homologues in the nuclear (host) genome. The
R. prowazekii genome contains the highest proportion of non-coding DNA
(24%) detected so far in a microbial genome. Such non-coding sequences may
be degraded remnants of 'neutralized' genes that await elimination
from the genome. Phylogenetic analyses indicate that R. prowazekii
is more closely related to mitochondria than is any other microbe studied
so far.
Mitochondrial DNA (mtDNA) sequences are widely used for inferring the phylogenetic relationships among species. Clearly, the
assumed model of nucleotide or amino acid substitution used should be as realistic as possible. Dependence among neighboring
nucleotides in a codon complicates modeling of nucleotide substitutions in protein-encoding genes. It seems preferable to
model amino acid substitution rather than nucleotide substitution. Therefore, we present a transition probability matrix of
the general reversible Markov model of amino acid substitution for mtDNA-encoded proteins. The matrix is estimated by the
maximum likelihood (ML) method from the complete sequence data of mtDNA from 20 vertebrate species. This matrix represents
the substitution pattern of the mtDNA-encoded proteins and shows some differences from the matrix estimated from the nuclear-encoded
proteins. The use of this matrix would be recommended in inferring trees from mtDNA-encoded protein sequences by the ML method.
The ability to recognize and repair abnormal DNA structures is common to all forms of life. Studies in a variety of species have identified an incredible diversity of DNA repair pathways. Documenting and characterizing the similarities and differences in repair between species has important value for understanding the origin and evolution of repair pathways as well as for improving our understanding of phenotypes affected by repair (e.g., mutation rates, lifespan, tumorigenesis, survival in extreme environments). Unfortunately, while repair processes have been studied in quite a few species, the ecological and evolutionary diversity of such studies has been limited. Complete genome sequences can provide potential sources of new information about repair in different species. In this paper, we present a global comparative analysis of DNA repair proteins and processes based upon the analysis of available complete genome sequences. We use a new form of analysis that combines genome sequence information and phylogenetic studies into a composite analysis we refer to as phylogenomics. We use this phylogenomic analysis to study the evolution of repair proteins and processes and to predict the repair phenotypes of those species for which we now know the complete genome sequence.
The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.
The ability to accurately predict gene function based on gene sequence is an important tool in many areas of biological research. Such predictions have become particularly important in the genomics age in which numerous gene sequences are generated with little or no accompanying experimentally determined functional information. Almost all functional prediction methods rely on the identification, characterization, and quantification of sequence similarity between the gene of interest and genes for which functional information is available. Because sequence is the prime determining factor of function, sequence similarity is taken to imply similarity of function. There is no doubt that this assumption is valid in most cases. However, sequence similarity does not ensure identical functions, and it is common for groups of genes that are similar in sequence to have diverse (although usually related) functions. Therefore, the identification of sequence similarity is frequently not enough to assign a predicted function to an uncharacterized gene; one must have a method of choosing among similar genes with different functions. In such cases, most functional prediction methods assign likely functions by quantifying the levels of similarity among genes. I suggest that functional predictions can be greatly improved by focusing on how the genes became similar in sequence (i.e., evolution) rather than on the sequence similarity itself. It is well established that many aspects of comparative biology can benefit from evolutionary studies (Felsenstein 1985), and comparative molecular biology is no exception
A versatile method, quartet puzzling, is introduced to reconstruct the topology (branching pattern) of a phylogenetic tree based on DNA or amino acid sequence data. This method applies maximum-likelihood tree reconstruction to all possible quartets that can be formed from n sequences. The quartet trees serve as starting points to reconstruct a set of optimal n- taxon trees. The majority rule consensus of these trees defines the quartet puzzling tree and shows groupings that are well supported. Computer simulations show that the performance of quartet puzzling to reconstruct the true tree is always equal to or better than that of neighbor joining. For some cases with high transition/transversion bias quartet puzzling outperforms neighbor joining by a factor of 10. The application of quartet puzzling to mitochondrial RNA and tRNA(Val) sequences from amniotes demonstrates the power of the approach. A PHYLIP-compatible ANSI C program, PUZZLE, for analyzing nucleotide or amino acid sequence data is available.
Human granulocytic ehrlichiosis (HGE) is a potentially fatal, tick-borne disease caused by a bacterium related or identical
toEhrlichia phagocytophila. To identify and characterizeE. phagocytophila group-specific protein antigen genes, we prepared and screened HGE agent and Ehrlichia equi genomic DNA expression libraries using polyclonal equine E. equiantibodies. Two clones, one each from HGE agent and E. equi, that were recognized specifically by antibodies to theE. phagocytophila group ehrlichiae had complete open reading frames of 3,693 and 3,615 nucleotides, respectively. The two clones were 96.6%
identical and predicted a protein with at least 11 tandemly repeated ankyrin motifs. Thus, the gene was namedank (for ankyrin). When the encoded protein, named AnkA, was expressed in Escherichia coli, it was recognized by antibodies from rabbits and mice immunized with the HGE agent, sera from humans convalescent from HGE,
and sera from horses convalescent from HGE and E. equi infection. Monospecific AnkA antibodies reacted with proteins in HGE agent immunoblots, and AnkA monoclonal antibodies detected
cytoplasmic antigen in E. phagocytophila group bacteria and also detected antigen associated with chromatin in infected but not uninfected HL-60 cell cultures. These
results suggest that this Ehrlichia protein may influence host cell gene expression.
We report a new transformation, the LogDet, that is consistent for sequences with differing nucleotide composition and that have arisen under simple but asymmetric stochastic models of evolution. This transformation is required because existing methods tend to group sequences on the basis of their nucleotide composition, irrespective of their evolutionary history. This effect of differing nucleotide frequencies is illustrated by using a tree-selection criterion on a simple distance measure defined solely on the basis of base composition, independent of the actual sequences. The new LogDet transformation uses determinants of the observed divergence matrices and works because multiplication of determinants (real numbers) is commutative, whereas multiplication of matrices is not,except in special symmetric cases. The use of determinants thus allows more general models of evolution with a symmetric rates of nucleotide change. The transformation is illustrated on a theoretical data set (where existing methods select the wrong tree) and with three biological data sets: chloroplasts, birds/mammals (nuclear), and honeybees ( mitochondrial ) . The LogDet transformation reinforces the logical distinction between transformations on the data and tree-selection criteria. The overall conclusions from this study are that irregular A,C,G,T compositions are an important and possible general cause of patterns that can mislead tree-reconstruction methods, even when high bootstrap values are obtained. Consequently, many published studies may need to be reexamined.
The heat-shock 60 proteins (Hsp60) constitute a subset of molecular chaperones essential for the survival of the cell, present in eubacteria as well as in eukaryotic organelles. Here, we have employed these highly conserved proteins for the inferences of the origins of the organelles. Hsp60s present in mitochondria from different eukaryotic lineages formed a clade, which showed the closest relationship to that of the Ehrlichia/Rickettsia cluster among the alpha-Proteobacteria. This, in addition to phenotypic characteristics, suggests that these obligate intracellular parasites and the lineage that generated the mitochondrion shared last common ancestry. In turn, Hsp60s present in chloroplasts from plants and a red alga, respectively, clustered specifically with those of the cyanobacteria, suggesting that all plastids derive exclusively from this eubacterial lineage.
Cytoplasmic incompatibility (CI) in Drosophila simulans is related to infection of the germ line by a rickettsial endosymbiont
(genus Wolbachia). Wolbachia were transferred by microinjection of egg cytoplasm into uninfected eggs of both D. simulans
and D. melanogaster to generate infected populations. Transinfected strains of D. melanogaster with lower densities of Wolbachia
than the naturally infected D. simulans strain did not express high levels of CI. However, transinfected D. melanogaster egg
cytoplasm, transferred back into D. simulans, generated infected populations that expressed CI at levels near those of the
naturally infected strain. A transinfected D. melanogaster line selected for increased levels of CI expression also displayed
increased symbiont densities. These data suggest that a threshold level of infection is required for normal expression of
CI and that host factors help determine the density of the symbiont in the host.
A continuous cell line, Aa23, was established from eggs of a strain of the Asian tiger mosquito, Aedes albopictus, naturally infected with the intracellular symbiont Wolbachia pipientis. The resulting cell line was shown to be persistently infected with the bacterial endosymbiont. Treatment with antibiotics cured the cells of the infection. In the course of establishing this cell line it was noticed that RFLPs in the PCR products of two Wolbachia genes from the parental mosquitoes were fixed in the infected cell line. This indicates that the mosquito host was naturally superinfected with different Wolbachia strains, whereas the infected cell line derived from these mosquitoes only contained one of the original Wolbachia strains. The development of an in vitro culture system for this fastidious microorganism should facilitate molecular analysis of the reproduction distorting phenotypes it induces in natural arthropod hosts.
Unfertilized eggs and fertilized embryos from Drosophila mothers mutant for the plutonium (plu) gene contain giant polyploid nuclei resulting from unregulated S-phase. The PLU protein, a 19-kDa ankyrin repeat protein, is present in oocytes and early embryos but is not detectable after the completion of the initial rapid S-M cycles of the embryo. The persistence of the protein during the early embryonic divisions is consistent with a direct role in linking S-phase and M-phase. When ectopically expressed in the eye disc, PLU did not perturb the cell cycle, suggesting that PLU regulates S-phase only in early embryonic development. The pan gu (png) and giant nuclei (gnu) genes also affect the S-phase in the unfertilized egg and early embryo. We show that functional png is needed for the presence of PLU protein. By analyzing png mutations of differing severity, we find that the extent of the png mutant phenotype inversely reflects the level of PLU protein. Our data suggest that PLU protein is required at the time of egg activation and the completion of meiosis.
Wolbachia are a group of intracellular inherited bacteria that infect a wide range of arthropods. They are associated with a number of different reproductive phenotypes in their hosts, such as cytoplasmic incompatibility, parthenogenesis and feminization. While it is known that the bacterial strains responsible for these different host phenotypes form a single clade within the alpha-Proteobacteria, until now it has not been possible to resolve the evolutionary relationships between different Wolbachia strains. To address this issue we have cloned and sequenced a gene encoding a surface protein of Wolbachia (wsp) from a representative sample of 28 Wolbachia strains. The sequences from this gene were highly variable and could be used to resolve the phylogenetic relationships of different Wolbachia strains. Based on the sequence of the wsp gene from different Wolbachia isolates we propose that the Wolbachia pipientis clade be initially divided into 12 groups. As more sequence information becomes available we expect the number of such groups to increase. In addition, we present a method of Wolbachia classification based on the use of group-specific wsp polymerase chain reaction (PGR) primers which will allow Wolbachia isolates to be typed without the need to clone and sequence individual Wolbachia genes. This system should facilitate future studies investigating the distribution and biology of Wolbachia strains from large samples of different host species.
The MutS protein of Escherichia coli plays a key role in the recognition and repair of errors made during the replication of DNA. Homologs of MutS have been found in many species including eukaryotes, Archaea and other bacteria, and together these proteins have been grouped into the MutS family. Although many of these proteins have similar activities to the E.coli MutS, there is significant diversity of function among the MutS family members. This diversity is even seen within species; many species encode multiple MutS homologs with distinct functions. To better characterize the MutS protein family, I have used a combination of phylogenetic reconstructions and analysis of complete genome sequences. This phylogenomic analysis is used to infer the evolutionary relationships among the MutS family members and to divide the family into subfamilies of orthologs. Analysis of the distribution of these orthologs in particular species and examination of the relationships within and between subfamilies is used to identify likely evolutionary events (e.g. gene duplications, lateral transfer and gene loss) in the history of the MutS family. In particular, evidence is presented that a gene duplication early in the evolution of life resulted in two main MutS lineages, one including proteins known to function in mismatch repair and the other including proteins known to function in chromosome segregation and crossing-over. The inferred evolutionary history of the MutS family is used to make predictions about some of the uncharacterized genes and species included in the analysis. For example, since function is generally conserved within subfamilies and lineages, it is proposed that the function of uncharacterized proteins can be predicted by their position in the MutS family tree. The uses of phylogenomic approaches to the study of genes and genomes are discussed.
Buchnera, the bacterial endosymbionts of aphids, undergo severe population bottlenecks during maternal transmission through their hosts. Previous studies suggest an increased effect of drift within these strictly asexual, small populations, resulting in an increased fixation of slightly deleterious mutations. This study further explores sequence evolution in Buchnera using three approaches. First, patterns of codon usage were compared across several homologous Escherichia coli and Buchnera loci, in order to test the prediction that selection for the use of optimal codons is less effective in small populations. A chi 2-based measure of codon bias was developed to adjust for the overall A + T richness of silent positions in the endosymbionts. In contrast to E. coli homologues, adaptive codon bias across Buchnera loci is markedly low, and patterns of codon usage lack a strong relationship with gene expression level. These data suggest that codon usage in Buchnera has been shaped largely by mutational pressure and drift rather than by selection for translational efficiency. One exception to the overall lack of bias is groEL, which is known to be constitutively overexpressed in Buchnera and other endosymbionts. Second, relative-rate tests show elevated rates of sequence evolution of numerous protein-coding loci across Buchnera, compared to E. coli. Finally, consistently higher ratios of nonsynonymous to synonymous substitutions in Buchnera loci relative to the enteric bacteria strongly suggest the accumulation of nonsynonymous substitutions in endosymbiont lineages. Combined, these results suggest a decreased effectiveness of purifying selection in purging endosymbiont populations of slightly deleterious mutations, particularly those affecting codon usage and amino acid identity.
The sequence of an alpha-proteobacterial genome, that of Rickettsia prowazekii, is a substantial advance in microbial and evolutionary biology. The genome of this obligately aerobic intracellular parasite is small and is apparently still undergoing reduction, reflecting gene losses attributable to its intracellular parasitic lifestyle. Evolutionary analyses of proteins encoded in the genome contain the strongest phylogenetic evidence to date for the view that mitochondria descend from alpha-proteobacteria. Although both Rickettsia and mitochondrial genomes are highly reduced, it appears that genome reduction in these lineages has occurred independently. Rickettsia's genome encodes an ATP-generating machinery that is strikingly similar to that of aerobic mitochondria. But it does not encode homologues for the ATP-producing pathways of anaerobic mitochondria or hydrogenosomes, leaving an important issue regarding the origin and nature of the ancestral mitochondrial symbiont unresolved.
The genome sequences of Chlamydia trachomatis mouse pneumonitis (MoPn) strain Nigg (1 069 412 nt) and Chlamydia pneumoniae strain AR39 (1 229 853 nt) were determined using a random shotgun strategy. The MoPn genome exhibited a general conservation of gene order and content with the previously sequenced C.trachomatis serovar D. Differences between C.trachomatis strains were focused on an approximately 50 kb 'plasticity zone' near the termination origins. In this region MoPn contained three copies of a novel gene encoding a >3000 amino acid toxin homologous to a predicted toxin from Escherichia coli O157:H7 but had apparently lost the tryptophan biosyntheis genes found in serovar D in this region. The C. pneumoniae AR39 chromosome was >99.9% identical to the previously sequenced C.pneumoniae CWL029 genome, however, comparative analysis identified an invertible DNA segment upstream of the uridine kinase gene which was in different orientations in the two genomes. AR39 also contained a novel 4524 nt circular single-stranded (ss)DNA bacteriophage, the first time a virus has been reported infecting C. pneumoniae. Although the chlamydial genomes were highly conserved, there were intriguing differences in key nucleotide salvage pathways: C.pneumoniae has a uridine kinase gene for dUTP production, MoPn has a uracil phosphororibosyl transferase, while C.trachomatis serovar D contains neither gene. Chromosomal comparison revealed that there had been multiple large inversion events since the species divergence of C.trachomatis and C.pneumoniae, apparently oriented around the axis of the origin of replication and the termination region. The striking synteny of the Chlamydia genomes and prevalence of tandemly duplicated genes are evidence of minimal chromosome rearrangement and foreign gene uptake, presumably owing to the ecological isolation of the obligate intracellular parasites. In the absence of genetic analysis, comparative genomics will continue to provide insight into the virulence mechanisms of these important human pathogens.
One of the main conclusions presented by the International Human Genome Sequencing Consortium is that ``hundreds of genes appear to have resulted from horizontal gene transfer from bacteria at some point in the vertebrate lineage''. We noticed that a significant proportion of these human genes have closely related orthologues in the primitive eukaryote Dictyostelium. This observation supports independent gene loss in multiple lineages (worm, fly, yeast, plants) rather than horizontal gene transfer from bacteria.
The members of the 10 kDa and 60 kDa heat-shock chaperonin proteins (Hsp10 and Hsp60 or Cpn10 and Cpn60), which form an operon in bacteria, are present in all eubacteria and eukaryotic ceil organelles such as mitochondria and chloroplasts. In archaebacteria and eukaryotic cell cytosol, no close homologues of Hsp10 or Hsp60 have been identified. However, these species (or ceil compartments) contain the Tcp-1 family of proteins (distant homologues of Hsp60). Phylogenetic analysis based on global alignments of Hsp60 and Hsp10 sequences presented here provide some evidence regarding the evolution of mitochondria from a member of the α-subdivision of Gram-negative bacteria and chloroplasts from cyanobacterial species, respectively. This inference is strengthened by the presence of sequence signatures that are uniquely shared between Hsp60 homologues from α-purple bacteria and mitochondria on one hand, and the chloroplasts and cyanobacterial hsp60s on the other. Within the α-purple subdivision, species such as Rickettsia and Ehrlichia, which live intracellularly within eukaryotic cells, are indicated to be the closest relatives of mitochondrial Homologues, In the Hsp60 evolutionary tree, rooted using the Tcp-1 homologue, the order of branching of the major groups was as follows: Gram-positive bacteria — cyanobacteria and chloroplasts — chlamydiae and spirochaetes —β and γ-Gram-negative purple bacteria —α-purple bacteria — mitochondria. A similar branching order was observed independently in the Hsp10 tree. Multiple Hsp60 homologues, when present in a group of species, were found to be clustered together in the trees, indicating that they evolved by independent gene-duplication events. This review also considers in detail the evolutionary relationship between Hsp50 and Tcp-1 families of proteins based on two different models (viz. archaebacterial and chimeric) for the origin of eukaryotic cell nucleus. Some predictions of the chimeric model are also discussed.
A new approach to assembling large, random shotgun sequencing projects has been developed. The TIGR Assembler overcomes several major obstacles to assembling such projects: the large number of pairwise comparisons required, the presence of repeat regions, chimeras introduced in the cloning process, and sequencing errors. A fast initial comparison of fragments based on oligonucleotide content is used to eliminate the need for a more sensitive comparison between most fragment pairs, thus greatly reducing computer search time. Potential repeat regions are recognized by determining which fragments have more potential overlaps than expected given a random distribution of fragments. Repeat regions are dealt with by increasing the match criteria stringency and by assembling these regions last so that maximum information from nonrepeat regions can be used. The algorithm also incorporates a number of constraints, such as clone length and the placement of sequences from the opposite ends of a clone. TIGR Assembler has been used to assemble the complete 1.8 Mbp Haemophilus influenzae (Fleischmann et al., 1995) and 0.58 Mbp Mycoplasma genitalium (Fraser et al., 1995) genomes.
The Organelle Genome Megasequencing Program (OGMP) investigates mitochondrial genome diversity and evolution by systematically determining the complete mitochondrial DNA (mtDNA) sequences of a phylogenetically broad selection of protists. The mtDNAs of lower fungi and choanoflagellates are being analyzed by the Fungal Mitochondrial Genome Project (FMGP), a sister project to the OGMP. Some of the most interesting protists include the jakobid flagellates Reclinomonas americana, Malawimonas jakobiformis, and Jakoba libera, which share ultrastructural similarities with amitochondriate retortamonads, and harbor mitochondrial genes not seen before in mtDNAs of other organisms. In R. americana and J. libera, gene clusters are found that resemble, to an unprecedented degree, the contiguous ribosomal protein operons str, S10, spc, and alpha of eubacteria. In addition, their mtDNAs code for an RNase P RNA that displays all the elements of a bacterial minimum consensus structure. This structure has been instrumental in detecting the rnpB gene in additional protists. Gene repertoire and gene order comparisons as well as multiple-gene phylogenies support the view of a single endosymbiotic origin of mitochondria, whose closest extant relatives are Rickettsia-type α-Proteobacteria.
We have conducted genome sequence analyses of seven prokaryotic microorganisms for which completely sequenced genomes are available (Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Mycoplasma genitalium, Synechocystis PCC6803 and Methanococcus jannaschii). We report the distribution of encoded known and putative polytopic cytoplasmic membrane transport proteins within these genomes. Transport systems for each organism were classified according to (1) putative membrane topology, (2) protein family, (3) bioenergetics, and (4) substrate specificities. The overall transport capabilities of each organism were thereby estimated. Probable function was assigned to greater than 90% of the putative transport proteins identified. The results show the following: (1) Numbers of transport systems in eubacteria are approximately proportional to genome size and correspond to 9.7 to 10.8% of the total encoded genes except for H. pylori (5.4%), Synechocystis (4.7%) and M. jannaschii (3.5%) which exhibit substantially lower proportions. (2) The distribution of topological types is similar in all seven organisms. (3) Transport systems belonging to 67 families were identified within the genomes of these organisms, and about half of these families are also found in eukaryotes. (4) 12% of these families are found exclusively in Gram-negative bacteria, but none is found exclusively in Gram-positive bacteria, cyanobacteria or archaea. (5) Two superfamilies, the ATP-binding cassette (ABC) and major facilitator (MF) superfamilies account for nearly 50% of all transporters in each organism, but the relative representation of these two transporter types varies over a tenfold range, depending on the organism. (6) Secondary, pmf-dependent carriers are 1.5 to threefold more prevalent than primary ATP-dependent carriers in E. coli, H. influenzae, H. pylori and B. subtilis while primary carriers are about twofold more prevalent in M. genitalium and Synechocystis. M. jannaschii exhibits a slight preference for secondary carriers. (7) Bioenergetics of transport generally correlate with the primary forms of energy generated via available metabolic pathways but ecological niche and substrate availability may also be determining factors. (8) All organisms display a similar range of transport specificities with quantitative differences presumably reflective of disparate ecological niches. (9) M. jannaschii and Synechocystis have a two to threefold increased proportion of transporters for inorganic ions with a concomitant decrease in transporters for organic compounds. (10) 6 to 18% of all transporters in these bacteria probably function as drug export systems showing that these systems are prevalent in non-pathogenic as well as pathogenic organisms. (11) All seven prokaryotes examined encode proteins homologous to known channel proteins, but none of the channel types identified occurs in all of these organisms. (12) The phosphoenolpyruvate:sugar phosphotransferase system is prevalent in the large genome organisms, E. coli and B. subtilis, and is present in the small genome organisms, H. influenzae and M. genitalium, but is totally lacking in H. pylori, Synechocystis and M. jannaschii. Details of the information summarized in this article are available on our web sites, and this information will be periodically updated and corrected as new sequence and biochemical data become available.
The vast majority of animals have intimate associations with bacteria that live inside them, sometimes as parasites, but often, as with gut flora, essential to their very existence. Recently, attention have been drawn to a class of bacteria that have profound implications for the evolution and ecology of the hosts they infect - bacteria that can manipulate their host's reproduction. So far, research has focused on invertebrates. This is the first comprehensive and systematic review of the activities of these reproductive parasites. It explores not only the intriguing theoretical questions raised by the relationships between symbiotic bacteria and their hosts, but also discusses a number of exciting practical applications, such as the potential for controlling disease-transmitting insects. Chapters on sex reversal, sex-ratio distortion, and evolution are included. This is a timely and up-to-the-minute exploration of a new, exciting, and rapidly expanding area of biology.
The CLUSTAL package of multiple sequence alignment programs has been completely rewritten and many new features added. The new software is a single program called CLUSTAL V, which is written in C and can be used on any machine with a standard C compiler. The main new features are the ability to store and reuse old alignments and the ability to calculate phylogenetic trees after alignment. The program is simple to use, completely menu driven and on-line help is provided.
Mutation frequency decline (MFD) is the rapid decrease in the frequency of certain induced nonsense suppressor mutations occurring when protein synthesis is transiently inhibited immediately after irradiation. MFD is abolished by mutations in the uvrA, -B, or -C genes, which prevent excision repair, or by a mfd mutation, which reduces the rate of excision but does not affect survival. Using an in vitro repair synthesis assay we found that although wild-type cells repair the transcribed (template) strand preferentially, mfd- cells are incapable of strand-specific repair. The deficiency in strand-selective repair of mfd- cell extract was corrected by adding highly purified "transcription-repair coupling factor" to the reaction mixture. We conclude that mfd is, most likely, the gene encoding the transcription-repair coupling factor.
Previous methods of heterogeneous ribonucleic acid (HnRNA) extraction yield material which "disaggregates" into small molecules. This could be the fault of either ribonuclease knicks in the polymers sustained during the extraction procedure or disaggregation into real subunits. The present communication distinguishes between these possibilities by describing an RNA extraction procedure which does not yield subunits when HnRNA is denatured. By the criteria of sedimentation through sucrose, formaldehyde, and dimethyl sulfoxide, it is estimated that the majority of the radioactivity of giant HnRNA after a 30-min pulse of [3H]uridine is associated with molecules in the range 5-10 × 106 daltons. In the electron microscope, under denaturing conditions, 84% (mass %) of giant HnRNA has a contour length of 4-9 μ corresponding to a molecular weight of about 5-10 × 106. Giant HnRNA has a "DNA-like" base composition (G + C = 46-54%) and has considerable secondary structure (ca. 60% helix conformation) as judged by its melting profile and reactivity with formaldehyde.
'Mutation frequency decline' (MFD) was discovered about forty years ago, and described as the disappearance of a particular class of ultraviolet light-induced mutations in Escherichia coli that occurred whenever protein synthesis was briefly inhibited immediately after irradiation. Later, MFD was interpreted as an excision repair anomaly uniquely affecting nonsense suppressor mutations induced in certain tRNA genes. Never fully understood, MFD has recently been linked to the newly discovered transcription-coupled rapid repair of ultraviolet damage on the template strand of active genes. This article recalls the emergence and development of the MFD story, and offers a new way to explain it and its relation to strand-specific excision repair.
Many bacteria live only within animal cells and infect hosts through cytoplasmic inheritance. These endosymbiotic lineages show distinctive population structure, with small population size and effectively no recombination. As a result, endosymbionts are expected to accumulate mildly deleterious mutations. If these constitute a substantial proportion of new mutations, endosymbionts will show (i) faster sequence evolution and (ii) a possible shift in base composition reflecting mutational bias. Analyses of 16S rDNA of five independently derived endosymbiont clades show, in every case, faster evolution in endosymbionts than in free-living relatives. For aphid endosymbionts (genus Buchnera), coding genes exhibit accelerated evolution and unusually low ratios of synonymous to nonsynonymous substitutions compared to ratios for the same genes for enterics. This concentration of the rate increase in nonsynonymous substitutions is expected under the hypothesis of increased fixation of deleterious mutations. Polypeptides for all Buchnera genes analyzed have accumulated amino acids with codon families rich in A+T, supporting the hypothesis that substitutions are deleterious in terms of polypeptide function. These observations are best explained as the result of Muller's ratchet within small asexual populations, combined with mutational bias. In light of this explanation, two observations reported earlier for Buchnera, the apparent loss of a repair gene and the overproduction of a chaperonin, may reflect compensatory evolution. An alternative hypothesis, involving selection on genomic base composition, is contradicted by the observation that the speedup is concentrated at nonsynonymous sites.
Existing approaches to sequencing the human genome are based on the assumption that each region to be sequenced must first be mapped. But there is a simpler strategy in which any number of laboratories can cooperate.
Although bacterial species display wide variation in their overall GC contents, the genes within a particular species' genome are relatively similar in base composition. As a result, sequences that are novel to a bacterial genome-i.e., DNA introduced through recent horizontal transfer-often bear unusual sequence characteristics and can be distinguished from ancestral DNA. At the time of introgression, horizontally transferred genes reflect the base composition of the donor genome; but, over time, these sequences will ameliorate to reflect the DNA composition of the new genome because the introgressed genes are subject to the same mutational processes affecting all genes in the recipient genome. This process of amelioration is evident in a large group of genes involved in host-cell invasion by enteric bacteria and can be modeled to predict the amount of time required after transfer for foreign DNA to resemble native DNA. Furthermore, models of amelioration can be used to estimate the time of introgression of foreign genes in a chromosome. Applying this approach to a 1.43-megabase continuous sequence, we have calculated that the entire Escherichia coli chromosome contains more than 600 kb of horizontally transferred, protein-coding DNA. Estimates of amelioration times indicate that this DNA has accumulated at a rate of 31 kb per million years, which is on the order of the amount of variant DNA introduced by point mutations. This rate predicts that the E. coli and Salmonella enterica lineages have each gained and lost more than 3 megabases of novel DNA since their divergence.
Mitochondria, organelles specialized in energy conservation reactions in eukaryotic cells, have evolved from eubacteria-like endosymbionts whose closest known relatives are the rickettsial group of alpha-proteobacteria. Because characterized mitochondrial genomes vary markedly in structure, it has been impossible to infer from them the initial form of the proto-mitochondrial genome. This would require the identification of minimally derived mitochondrial DNAs that better reflect the ancestral state. Here we describe such a primitive mitochondrial genome, in the freshwater protozoon Reclinomonas americana. This protist displays ultrastructural characteristics that ally it with the retortamonads, a protozoan group that lacks mitochondria. R. americana mtDNA (69,034 base pairs) contains the largest collection of genes (97) so far identified in any mtDNA, including genes for 5S ribosomal RNA, the RNA component of RNase P, and at least 18 proteins not previously known to be encoded in mitochondria. Most surprising are four genes specifying a multisubunit, eubacterial-type RNA polymerase. Features of gene content together with eubacterial characteristics of genome organization and expression not found before in mitochondrial genomes indicate that R. americana mtDNA more closely resembles the ancestral proto-mitochondrial genome than any other mtDNA investigated to date.
This paper describes a new system, GLIMMER, for finding genes in microbial genomes. In a series of tests on Haemophilus influenzae, Helicobacter pylori and other complete microbial genomes, this system has proven to be very accurate at locating virtually all the genes in these
sequences, outperforming previous methods. A conservative estimate based on experiments on H.pylori and H.influenzae is that the system finds >97% of all genes. GLIMMER uses interpolated Markov models (IMMs) as a framework for capturing dependencies
between nearby nucleotides in a DNA sequence. An IMM-based method makes predictions based on a variable context; i.e., a variable-length
oligomer in a DNA sequence. The context used by GLIMMER changes depending on the local composition of the sequence. As a result,
GLIMMER is more flexible and more powerful than fixed-order Markov methods, which have previously been the primary content-based
technique for finding genes in microbial DNA.
The availability of the complete sequence of Escherichia coli strain MG1655 provides the first opportunity to assess the overall impact of horizontal genetic transfer on the evolution of bacterial genomes. We found that 755 of 4,288 ORFs (547.8 kb) have been introduced into the E. coli genome in at least 234 lateral transfer events since this species diverged from the Salmonella lineage 100 million years (Myr) ago. The average age of introduced genes was 14.4 Myr, yielding a rate of transfer 16 kb/Myr/lineage since divergence. Although most of the acquired genes subsequently were deleted, the sequences that have persisted ( approximately 18% of the current chromosome) have conferred properties permitting E. coli to explore otherwise unreachable ecological niches.
We have cloned and sequenced the genes encoding cytochrome b (cob) and cytochrome c oxidase subunit I (cox1) from Rickettsia prowazekii, a member of the alpha-proteobacteria. The phylogenetic analysis supports the hypothesis that mitochondria are derived from the alpha-proteobacteria and more specifically from within the Rickettsiaceae. We have estimated that the common ancestor of mitochondria and Rickettsiaceae dates back to more than 1500 million years ago.
In a recent analysis J.G. Lawrence and H. Ochman [Proc Natl Acad Sci USA 1998;95:9413-9417 (Reference 1)] surmised that about 10% of the current E. coli genome consists of genes that were acquired in over 200 events of lateral gene transfer, which occurred subsequent to the divergence of E. coli and Salmonella some 100 million years ago. Overall, the data suggest that no less than 18% of E. coli's genes might be relatively recent foreign acquisitions, and that the average rate of acquisition may be close to about 16 kb per million years. These quantitative estimates of comparatively recent genome flux have profound impact on evolutionary genome comparisons. They tend to suggest that a search should be on to identify principles that might ultimately govern gene distribution patterns across prokaryotic genomes.
A new system for aligning whole genome sequences is described. Using an efficient data structure called a suffix tree, the
system is able to rapidly align sequences containing millions of nucleotides. Its use is demonstrated on two strains of Mycobacterium tuberculosis, on two less similar species of Mycoplasma bacteria and on two syntenic sequences from human chromosome 12 and mouse chromosome
6. In each case it found an alignment of the input sequences, using between 30 s and 2 min of computation time. From the system
output, information on single nucleotide changes, translocations and homologous genes can easily be extracted. Use of the
algorithm should facilitate analysis of syntenic chromosomal regions, strain-to-strain comparisons, evolutionary comparisons
and genomic duplications.
A software tool was implemented that computes exact repeats and palindromes in entire genomes very efficiently.
Availability:
Via the Bielefeld Bioinformatics Server (http://bibiserv.techfak.uni-bielefeld.de/rep uter/).
A new method has been developed for rapidly closing a large number of gaps in a whole-genome shotgun sequencing project. The method employs multiplex PCR and a novel pooling strategy to minimize the number of laboratory procedures required to sequence the unknown DNA that falls in between contiguous sequences. Multiplex sequencing, a novel procedure in which multiple PCR primers are used in a single sequencing reaction, is used to interpret the multiplex PCR results. Two protocols are presented, one that minimizes pipetting and another that minimizes the number of reactions. The pipette optimized multiplex PCR method has been employed in the final phases of closing the Streptococcus pneumoniae genome sequence, with excellent results.