An attempted transinfection of Wolbachia in the Western honey bee (Apis mellifera)

<p>Wolbachia, an intracellular endosymbiont found in up to 60% of arthropods, has been celebrated for its highly varied host-phenotype interactions. These effects are diverse, ranging from reproductive manipulations to obligate mutualisms and facultative symbiosis. These facultative effects include increased resistance to, and reduction in the ability to vector, a number of RNA viruses in insects. Artificial transinfection to mediate human vector-borne diseases such as Dengue fever and Zika virus in Aedes mosquitoes has had considerable success globally. However, using Wolbachia to mediate zoonotic disease directly in threatened species has not been examined. The Western honey bee (Apis mellifera) has shown significant global population declines across the US and Europe, suffering from a diverse range of pathogens, including viral RNA and parasite vector networks. Wolbachia infection in honey bees has only been detected once and its effects have not been investigated. Here, I present the first attempted transinfection of Wolbachia in the Western honey bee using established transinfection protocols. The natural, but rarely found, Wolbachia infection reported in A. mellifera was examined against a robust phylogeny of all existing Wolbachia supergroups, a feat that has not been updated in the literature since 2015. I discovered Wolbachia infection in Ancistrocerus gazella, the European tube wasp, where it has never been observed. I isolated the natural Wolbachia strain hosted by Drosophila melanogaster (wMel ) and more than 1200 individuals from a range of honey bee life stages (from eggs to adults) were used as potential Wolbachia recipients using sound microinjection protocols. Additionally, I present a novel transinfection avenue utilizing artificial insemination and honey bee breeding using Wolbachia-inoculated drone semen. When no individuals were successfully infected with Wolbachia in F0 or F1, I investigated the expression of several antimicrobial peptides to characterize the immune response in young larvae to Wolbachia microinjection. There was a significant upregulation of apidaecin when injected with live Wolbachia, but not heat-treated bacteria, which has never been reported in host immune response to Wolbachia previously. The findings presented in this study highlight the importance of Wolbachia strain selection, immune response to Wolbachia, and the potential requirement for cell line culture in future transinfection attempts into A. mellifera. These findings will help inform future transinfection attempts, which are encouraged.</p>

An attempted transinfection of Wolbachia in the Western honey bee (Apis mellifera)

<p>Wolbachia, an intracellular endosymbiont found in up to 60% of arthropods, has been celebrated for its highly varied host-phenotype interactions. These effects are diverse, ranging from reproductive manipulations to obligate mutualisms and facultative symbiosis. These facultative effects include increased resistance to, and reduction in the ability to vector, a number of RNA viruses in insects. Artificial transinfection to mediate human vector-borne diseases such as Dengue fever and Zika virus in Aedes mosquitoes has had considerable success globally. However, using Wolbachia to mediate zoonotic disease directly in threatened species has not been examined. The Western honey bee (Apis mellifera) has shown significant global population declines across the US and Europe, suffering from a diverse range of pathogens, including viral RNA and parasite vector networks. Wolbachia infection in honey bees has only been detected once and its effects have not been investigated. Here, I present the first attempted transinfection of Wolbachia in the Western honey bee using established transinfection protocols. The natural, but rarely found, Wolbachia infection reported in A. mellifera was examined against a robust phylogeny of all existing Wolbachia supergroups, a feat that has not been updated in the literature since 2015. I discovered Wolbachia infection in Ancistrocerus gazella, the European tube wasp, where it has never been observed. I isolated the natural Wolbachia strain hosted by Drosophila melanogaster (wMel ) and more than 1200 individuals from a range of honey bee life stages (from eggs to adults) were used as potential Wolbachia recipients using sound microinjection protocols. Additionally, I present a novel transinfection avenue utilizing artificial insemination and honey bee breeding using Wolbachia-inoculated drone semen. When no individuals were successfully infected with Wolbachia in F0 or F1, I investigated the expression of several antimicrobial peptides to characterize the immune response in young larvae to Wolbachia microinjection. There was a significant upregulation of apidaecin when injected with live Wolbachia, but not heat-treated bacteria, which has never been reported in host immune response to Wolbachia previously. The findings presented in this study highlight the importance of Wolbachia strain selection, immune response to Wolbachia, and the potential requirement for cell line culture in future transinfection attempts into A. mellifera. These findings will help inform future transinfection attempts, which are encouraged.</p>

The Incidence of Wolbachia Bacterial Endosymbiont in Bisexual and Parthenogenetic Populations of the Psyllid Genus Cacopsylla (Hemiptera, Psylloidea)

Wolbachia is one of the most common intracellular bacteria; it infects a wide variety of insects, other arthropods, and some nematodes. Wolbachia is ordinarily transmitted vertically from mother to offspring and can manipulate physiology and reproduction of their hosts in different ways, e.g., induce feminization, male killing, and parthenogenesis. Despite the great interest in Wolbachia, many aspects of its biology remain unclear and its incidence across many insect orders, including Hemiptera, is still poorly understood. In this report, we present data on Wolbachia infection in five jumping plant-lice species (Hemiptera, Psylloidea) of the genus Cacopsylla Ossiannilsson, 1970 with different reproductive strategies and test the hypothesis that Wolbachia mediates parthenogenetic and bisexual patterns observed in some Cacopsylla species. We show that the five species studied are infected with a single Wolbachia strain, belonging to the supergroup B. This strain has also been found in different insect orders (Lepidoptera, Hemiptera, Plecoptera, Orthoptera, Hymenoptera, Diptera) and even in acariform mites (Trombidiformes), suggesting extensive horizontal transmission of Wolbachia between representatives of these taxa. Our survey did not reveal significant differences in infection frequency between parthenogenetic and bisexual populations or between males and females within bisexual populations. However, infection rate varied notably in different Cacopsylla species or within distinct populations of the same species. Overall, we demonstrate that Wolbachia infects a high proportion of Cacopsylla individuals and populations, suggesting the essential role of this bacterium in their biology.

New Strains of Wolbachia Unveiling the Complexity of This Symbiotic Interaction in Solenopsis (Hymenoptera: Formicidae)

Bacteria of the genus Wolbachia are widely distributed in arthropods, particularly in ants; nevertheless, it is still little explored with the Multilocus Sequence Typing (MLST) methodology, especially in the genus Solenopsis, which includes species native to South America. Ants from this genus have species distributed in a cosmopolitan way with some of them being native to South America. In Brazil, they are widely spread and preferentially associated with areas of human activity. This study aimed to investigate the diversity of Wolbachia in ants of the genus Solenopsis through the MLST approach and their phylogenetic relationship, including the relationship between mtDNA from the host and the related Wolbachia strain. We also tested the geographic correlation between the strains to infer transmission and distributional patterns. Fifteen new strains and eleven previously unknown alleles were obtained by sequencing and analyzing the five genes that make up the MLST. The phylogenetic relationship between the strains showed a polyphyletic pattern, indicative of the complexity of the evolutionary history of these bacteria in the analyzed species. We detected the correlation of host’s mitochondrial DNA with Wolbachia diversity which imply that related strains exist in related hosts, strongly suggesting the occurrence of vertical transfer. We found no specificity of the Wolbachia strain for a given geographic region that could indicate either that there is no horizontal transfer of the strain from the environment for the host or that the human action could be shuffling the distribution of the Solenopsis ants and the endosymbiont Wolbachia, as well. Our study highlights the complexity and novelty of Wolbachia diversity with this specific group of ants and the need for further studies that focus on understanding of this intricate relationship.

Near-Complete Genome Sequences of a Wolbachia Strain Isolated from Diaphorina citri Kuwayama (Hemiptera: Liviidae)

ABSTRACT Wolbachia strains are one of three endosymbionts associated with the insect vector of “Candidatus Liberibacter asiaticus,” Diaphorina citri Kuwayama (Hemiptera: Liviidae). We report three near-complete genome sequences of samples of Wolbachia from D. citri (wDi), with sizes of 1,518,595, 1,542,468, and 1,538,523 bp.

Isolation and Propagation of Laboratory Strains and a Novel Flea-Derived Field Strain of Wolbachia in Tick Cell Lines

Wolbachia are intracellular endosymbionts of several invertebrate taxa, including insects and nematodes. Although Wolbachia DNA has been detected in ticks, its presence is generally associated with parasitism by insects. To determine whether or not Wolbachia can infect and grow in tick cells, cell lines from three tick species, Ixodes scapularis, Ixodes ricinus and Rhipicephalus microplus, were inoculated with Wolbachia strains wStri and wAlbB isolated from mosquito cell lines. Homogenates prepared from fleas collected from cats in Malaysia were inoculated into an I. scapularis cell line. Bacterial growth and identity were monitored by microscopy and PCR amplification and sequencing of fragments of Wolbachia genes. The wStri strain infected Ixodes spp. cells and was maintained through 29 passages. The wAlbB strain successfully infected Ixodes spp. and R. microplus cells and was maintained through 2–5 passages. A novel strain of Wolbachia belonging to the supergroup F, designated wCfeF, was isolated in I. scapularis cells from a pool of Ctenocephalides sp. cat fleas and maintained in vitro through two passages over nine months. This is the first confirmed isolation of a Wolbachia strain from a flea and the first isolation of any Wolbachia strain outside the “pandemic” A and B supergroups. The study demonstrates that tick cells can host multiple Wolbachia strains, and can be added to panels of insect cell lines to improve success rates in isolation of field strains of Wolbachia.

Isolation and Propagation of Laboratory Strains and a Novel Flea-Derived Field Strain of Wolbachia in Tick Cell Lines

Wolbachia are intracellular endosymbionts of several invertebrate taxa, including insects and nematodes. Although Wolbachia DNA has been detected in ticks, its presence is generally associated with parasitism by insects. To determine whether or not Wolbachia can infect and grow in tick cells, cell lines from three tick species, Ixodes scapularis, Ixodes ricinus and Rhipicephalus microplus, were inoculated with Wolbachia strains wStri and wAlbB isolated from mosquito cell lines. Homogenates prepared from fleas collected from cats in Malaysia were inoculated into an I. scapularis cell line. Bacterial growth and identity were monitored by microscopy and PCR amplification and sequencing of fragments of Wolbachia genes. The wStri strain infected Ixodes spp. cells and was maintained through 29 passages. The wAlbB strain successfully infected Ixodes spp. and R. microplus cells and was maintained through 2-5 passages. A novel strain of Wolbachia belonging to the supergroup F, designated wCfeF, was isolated in I. scapularis cells from a pool of Ctenocephalides sp. cat fleas and maintained in vitro through two passages over nine months. This is the first confirmed isolation of a Wolbachia strain from a flea and the first isolation of any Wolbachia strain outside the &ldquo;pandemic&rdquo; A and B supergroups. The study demonstrates that tick cells can host multiple Wolbachia strains, and can be added to panels of insect cell lines to improve success rates in isolation of field strains of Wolbachia.