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An analysis of Wolbachia incidence and genetics in non-ant Hymenoptera diversity.

Wolbachia bacteria are widespread maternally inherited symbionts of Nematoda and diverse Arthropoda hosts. Their evolutionary success is determined by the ability to affect the biology of the host in different ways, promoting the relative fitness of females harbouring Wolbachia, as well as sporadic cases of horizontal transmission of Wolbachia between different host species. Here, we revised Wolbachia infection in the Hymenoptera with respect to the symbiont occurrence in host taxa and Wolbachia genetics. The representatives of about half of the extant families and 1000 out of 140,000 non-ant hymenopteran species have been tested for Wolbachia infection. We concluded that Wolbachia are found in all major hymenopteran families. More than 75% of Wolbachia diversity belongs to the A supergroup, whereas other variants belong to the B supergroup and only two isolates belong to the supergroup F. One of the main results of this study is the discovery of a specific Wolbachia genetic pattern (based on multilocus sequence typing [MLST]) in Apoidea hosts. Two haplotypes, ST-479 and ST-wH14, along with their alleles within other sequence types (STs), form the core of symbiont diversity, comprising 81% of unique host-Wolbachia ST associations. These haplotypes have not been reported beyond the Apoidea superfamily or Hymenoptera order. The reasons and mechanisms underlying this pattern in Apoidea remain unknown. Another important result of our study concerns the use of the MLST protocol, which has been previously criticised. We analysed 51 Wolbachia genomes for the average nucleotide identity (ANI) and MLST data, and found that genome and MLST variation are highly correlated. Therefore, the MLST protocol for Wolbachia remains reliable for many research tasks.

Animals

Wolbachia Host Shifts and Widespread Occurrence of Reproductive Manipulation Loci in European Butterflies.

Wolbachia is the most frequent bacterial endosymbiont of arthropods and nematodes. Although it is mostly vertically transmitted, from parent to offspring through the egg cytoplasm, horizontal transfer of Wolbachia is thought to be common over evolutionary timescales. However, the relative frequency of each transmission mechanism has not been studied systematically in closely related species. Additionally, while Wolbachia is generally regarded as a reproductive manipulator, it is unclear how frequently the symbiont induces such effects. In this study, we investigated the presence, phenotypes and phylogenetic relationships among Wolbachia strains in whole genome sequence data for 18 European butterfly sister-species pairs. We find that sister-species share Wolbachia strains more often than random species pairs and that the probability of strain sharing is higher for younger pairs of host species, especially those with greater range overlap. We also find that split times between Wolbachia strains that infect the same sister-species pair generally pre-date host divergence, ruling out co-divergence in favour of horizontal transfer. However, some strains are younger than the mitochondrial split times of their hosts, so introgressive transfer cannot be ruled out in some cases. In addition, all newly assembled Wolbachia genomes contained putative homologues of genes associated with cytoplasmic incompatibility and male killing. This supports the potential for reproductive manipulation in Wolbachia strains infecting European butterflies, which until now was only inferred from mitochondrial diversity patterns. Our results show that horizontal and introgressive transfer of Wolbachia are frequent even between recently speciated host taxa, suggesting the symbiont's turnover rate is higher than had been inferred previously from surveys of distantly related hosts.

Animals

Wolbachia strain wLhui induces temperature-dependent incomplete cytoplasmic incompatibility in the invasive pest Liriomyza huidobrensis with biocontrol potential.

BACKGROUND: Wolbachia is a maternally inherited endosymbiont that manipulates host reproduction through cytoplasmic incompatibility (CI), offering promising opportunities for biocontrol of agricultural pests. The leaf-miner Liriomyza huidobrensis (Blanchard) is a globally invasive and highly polyphagous pest with a high incidence of Wolbachia infection; however, its reproductive effects remain poorly understood. Here, we investigated the reproductive manipulation induced by the Wolbachia strain wLhui using genomics analyses and crossing assays. RESULTS: wLhui localized primarily to the reproductive tissues of both female and male adults and maintained a 100% infection prevalence across three host generations under both low (15 and 20 °C) and moderate (25 °C) temperatures. Crossing assays showed that wLhui induced incomplete CI, reducing egg hatch by approximately 30% in incompatible crosses. Both CI strength (sh) and wLhui density varied with host rearing temperature. Genome sequencing revealed that wLhui (approximately 1.27 Mb) belongs to supergroup A and harbors two pairs of CI factor genes (cifA and cifB). These Cif proteins are classified as Type I and exhibit substantial phylogenetic and structural divergence. Expression of CifB-pair1 caused growth defects in yeast, suggesting that CifB-pair1 exhibits toxicity. However, no direct interaction between CifA and CifB was detected by yeast two-hybrid assays. CONCLUSIONS: These findings elucidate the role and molecular basis of wLhui-induced reproductive manipulation and highlight its potential for developing Wolbachia-based biocontrol strategies against leaf-miner pests. © 2026 Society of Chemical Industry.

Animals

Remodeling of host lipid metabolism by Wolbachia strain wAlbB is associated with lipid accumulation and cardiolipin dysregulation in the Aedes aegypti fat body.

BACKGROUND: The intracellular symbiont Wolbachia, particularly the wAlbB strain, is a promising biocontrol agent against mosquito-borne diseases. Although Wolbachia infection is known to perturb host metabolism, the underlying mechanisms, especially those related to lipid metabolism, remain poorly understood. METHODS: We performed an integrated multi-level analysis of the Aedes aegypti fat body in uninfected and wAlbB-infected mosquitoes, combining histology, biochemistry, untargeted liquid chromatography-mass spectrometry (LC-MS) lipidomics, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis, reverse transcription quantitative PCR of key metabolic genes, and quantification of acetyl-coenzyme A (acetyl-CoA) and reduced nicotinamide adenine dinucleotide (NADH) levels. RESULTS: wAlbB infection increased fat body wet weight and thickness, accompanied by accumulation of triglyceride and of lipid droplets. Lipidomic analysis further revealed extensive lipidome remodeling, with elevated free fatty acid, diglyceride, and triglyceride, but broad depletion of glycerophospholipids, particularly cardiolipin. These changes were supported by transcriptional alterations: upregulation of fatty acid synthase 1 and glycerol-3-phosphate acyltransferase 1, and downregulation of adipose triglyceride lipase and carnitine palmitoyltransferase 1. Cardiolipin depletion correlated with downregulation of genes involved in its synthesis and remodeling, including phosphatidylglycerophosphate synthase and calcium-independent phospholipase A2γ. These lipid changes were also associated with accumulation of acetyl-CoA and NADH. CONCLUSIONS: Our findings suggest that wAlbB infection is associated with extensive lipid metabolic remodeling in the Aedes aegypti fat body, characterized by accumulation of neutral lipids and cardiolipin depletion, accompanied by transcriptional remodeling of key metabolic enzymes. This study establishes the fat body as a primary tissue-level hub for Wolbachia-associated lipid remodeling and provides a foundational framework for future mechanistic investigations into host-symbiont metabolic interactions.

Animals

Isolation of spotted fever group and Wolbachia-like agents from field-collected materials by means of plaque formation in mammalian and mosquito cells.

Three isolations from ticks (Dermacentor occidentalis) of a rickettsia of the spotted fever group and 5 isolations from chipmunk (Eutamias rugicaudus) blood of a Wolbachia-like agent were obtained from plaques formed in Singh's Aedes albopictus (mosquito) and Vero (African green monkey kidney) cell cultures. These organisms could not be isolated by injection of the infected ticks or blood into embryonated chicken eggs, guinea pigs, or voles (Microtus pennsylvanicus), but fluid cultures of Grace's Antheraea eucalypti (moth1 and Singh's A. albopictus cells inoculated with the bloods yielded the Wolbachia-like agent.

Aedes

[Immunologic comparison between some Wolbachiae and research of antigentic community with other members of the order of Rickettsiales].

The antigenic relationship between some rickettsiae of arthropods, which do not transmit germs pathogenic to vertebrates was investigated by agglutination and immunofluorescence techniques. The comparison of R. melolonthae, R. tipulae, R. cetonidarum, R. grylli and of the rickettsiae of the scorpion Buthus occitanus shows that the Wolbachiae are divided into three serological groups. The insect crystallogen rickettsiae belong to the first group, R. grylli and the scorpion Rickettsia are antigenically distinct (tables I and II). No antigenic relationship was found between these arthropod microorganisms and the following agents of vertebrate rickettsioses and chlamydioses : historical typhus, murine typhus, pustulous fever, Q-fever, epizootic absortion of sheep and psittacosis (tables III and IV). Bacteria of OX2 and OX19 strain of P. vulgaris when mixed with anti-Wolbachiae serums of I, II, III groups, give a negative Weil a Felix reaction. The OXK Proteus are however agglutinated by 4 out of 6 rabbit anti-Rickettsiella serums of the group. This fact calls for a further investigation regarding the comparison of these Rickettsiella with various strains of R. tsutsugamushi.

Antibodies, Bacterial

Molecular identification of Wolbachia, the agent of cytoplasmic incompatibility in Drosophila simulans, and variability in relation with host mitochondrial types.

Sequences of a segment of the 16S ribosomal DNA of Wolbachia, a rickettsia-like microorganism responsible for cytoplasmic incompatibility in Drosophila simulans, have been obtained after polymerase chain reaction (PCR) amplification. Their comparison with other eubacterial sequences allows us to assign these endosymbionts to the alpha subdivision of purple bacteria. Four related sequences have been obtained for microorganisms carried by eight isofemale lines representative of the three mitochondrial types of D. simulans. Their phylogeny and level of divergence do not parallel that of the mitochondrial DNA, suggesting that several independent infections occurred. There is no direct relation between bacterial phylogeny and formerly identified incompatibility types.

Animals

Wolbachia endosymbionts responsible for various alterations of sexuality in arthropods.

Rickettsia-like maternally inherited bacteria have been shown to be involved in a variety of alterations of arthropod sexuality, such as female-biased sex ratios, parthenogenesis, and sterility of crosses either between infected males and uninfected females or between infected individuals (cytoplasmic incompatibility). We have characterized several of these microorganisms through partial sequences of the small (16S) and large (23S) subunit ribosomal DNA. All the symbionts identified, which include several cytoplasmic incompatibility microorganisms, several endosymbionts of terrestrial isopods, and symbionts of two thelytokous Trichogramma wasp species, belong to a monophyletic group of related symbionts, some of which have previously been detected in several insects exhibiting cytoplasmic incompatibility. Three molecular lineages can be identified on the basis of 16S as well as 23S sequences. Although they are only known as endocellular symbionts, Wolbachia spread by horizontal transfer across host lineages as evidenced by their diversification which occurred long after that of their hosts, and by the non-congruence of the phylogenetic relationships of symbionts and their hosts. Indeed, symbionts of two different lineages have been found in the same host species, whereas closely related endosymbionts are found in distinct insect orders. Isopod endosymbionts form a separate lineage, and they can determine feminization as well as cytoplasmic incompatibility. The ability to determine cytoplasmic incompatibility, found in all lineages, is probably ancestral to this group.

Animals

Evolutionary Diversification and Functions of the Candidate Male Killing Gene wmk.

Symbiont-mediated male killing (MK) is a mechanism that selectively eliminates male offspring, often by disrupting sex-specific developmental processes. In Drosophila melanogaster, the WO-mediated killing gene wmk from Wolbachia prophage WO transgenically reproduces the MK phenotype, yet how the gene evolves and functions across diverse Wolbachia has not been systematically investigated. We analyzed 32 Wolbachia genomes available in the NCBI database to study wmk homologs across different arthropod hosts, reproductive parasitism functions, and Wolbachia supergroups. First, we report at least five distinct wmk phylogenetic clusters (Types I to V), often organized in multigenic dyads or triads. Second, among MK Wolbachia, there is a significantly higher number of wmk genes and diversity in Lepidoptera strains than in Drosophila strains, which exclusively harbor wmk Types I and III. Third, there are three patterns of wmk sequence and genomic organizational changes in Drosophila MK strains that associate with different evolutionary trajectories underpinning the MK phenotype. Fourth, single and combinatory transgenic expression of Types I and III in D. melanogaster uncovers male-biased lethality associated with Type I; however, dual expression of the Types together elicits a major reduction in offspring number. Fifth, wmk genes have low expression level across D. melanogaster developmental stages relative to the cifA and cifB genes, which could explain why cytoplasmic incompatibility is expressed in this system. These findings establish a complex and phylogenetically informed genetic basis of wmk-induced lethality, highlighting the role of gene copy number and expression, wmk Types, and host background in shaping the phenotype.

Animals

16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects.

Bacterial endosymbionts of insects have long been implicated in the phenomenon of cytoplasmic incompatibility, in which certain crosses between symbiont-infected individuals lead to embryonic death or sex ratio distortion. The taxonomic position of these bacteria has, however, not been known with any certainty. Similarly, the relatedness of the bacteria infecting various insect hosts has been unclear. The inability to grow these bacteria on defined cell-free medium has been the major factor underlying these uncertainties. We circumvented this problem by selective PCR amplification and subsequent sequencing of the symbiont 16S rRNA genes directly from infected insect tissue. Maximum parsimony analysis of these sequences indicates that the symbionts belong in the alpha-subdivision of the Proteobacteria, where they are most closely related to the Rickettsia and their relatives. They are all closely related to each other and are assigned to the type species Wolbachia pipientis. Lack of congruence between the phylogeny of the symbionts and their insect hosts suggest that horizontal transfer of symbionts between insect species may occur. Comparison of the sequences for W. pipientis and for Wolbachia persica, an endosymbiont of ticks, shows that the genus Wolbachia is polyphyletic. A PCR assay based on 16S primers was designed for the detection of W. pipientis in insect tissue, and initial screening of insects indicates that cytoplasmic incompatibility may be a more general phenomenon in insects than is currently recognized.

Animals

Transovarial transmission of Rickettsia-like microorganisms in mosquitoes.

The wolbachiae found in Culex pipiens and the Tafahi strain of the A. scutellaris group are small rickettsia-like symbionts of the gonads. They are extrachromosomal self-replicating units that are vertically transmitted through the ovaries. Their presence in the only two groups of mosquitoes known to exhibit incompatibility, the fact that they are found in only the Tafahi strain, and the loss of incompatibility after removal of Wolbachia in C. pipiens are compelling evidence for the role that Wolbachia plays in incompatibility.

Aedes

Piscirickettsia salmonis gen. nov., sp. nov., the causative agent of an epizootic disease in salmonid fishes.

A novel intracellular pathogen morphologically similar to the ehrlichiae has been isolated in cell culture and identified as the cause of an epizootic disease of salmonid fish. Like the ehrlichiae, the salmonid pathogen, designated strain LF-89, replicates within membrane-bound cytoplasmic vacuoles in host cells. This agent is the first with characteristics of this type to be isolated from a fish. Analysis of the LF-89 16S rRNA indicated that, unlike the ehrlichiae, LF-89 is a gamma proteobacterium distantly related to Coxiella burnetii and perhaps Wolbachia persica. A new genus and species (Piscirickettsia salmonis gen. nov., sp. nov.) are proposed for this organism, with ATCC(R) VR 1361 as the type strain.

Animals

The ecology, evolution, and physiology of Cardinium: a widespread heritable endosymbiont of invertebrates.

Candidatus Cardinium hertigii (Cardinium) are maternally transmitted obligate intracellular bacteria found in a wide range of invertebrate hosts, including arthropods and nematodes. Infection with Cardinium has substantial consequences for host biology, with many strains manipulating host reproduction to favor symbiont transmission by (i) feminizing male hosts, (ii) altering host sex allocation, (iii) inducing parthenogenesis, or (iv) causing cytoplasmic incompatibility. Other Cardinium strains can confer benefits to their host or alter host behavior. Cardinium-modified host phenotypes can result in selective sweeps of cytological elements through host populations and potentially reinforce host speciation. Cardinium has potential for applications in controlling arthropod pest species and arthropod-vectored disease transmission, although much remains to be explored regarding Cardinium physiology and host interactions. In this review, we provide an overview of Cardinium evolution and host distribution. We describe the various host phenotypes associated with Cardinium and how biological and environmental factors influence these symbioses. We also provide an overview of Cardinium metabolism, physiology, and potential mechanisms for interactions with hosts based on recent studies using genomics and transcriptomics. Finally, we discuss new methodologies and directions for Cardinium research, including improving our understanding of Cardinium physiology, response to environmental stress, and potential for controlling arthropod pest populations.

Symbiosis