Evolution ofWolbachiaMutualism and Reproductive Parasitism: Insight from Two Novel Strains that Co-infect Cat Fleas

Abstract

Importance 44Many arthropod and certain nematode species are infected with wolbachiae which are 45 intracellular bacteria well known for reproductive parasitism (RP). Like other RP 46 strategies, Wolbachia-induced cytoplasmic incompatibility, CI, increases prevalence and 47 frequency in host populations. Mutualism is another strategy employed by wolbachiae 48 to maintain host infection, with some strains synthesizing and supplementing certain B 49 vitamins (particularly biotin) to invertebrate hosts. Curiously, we discovered two novel 50Wolbachia strains that co-infect cat fleas (Ctenocephalides felis): wCfeT carries biotin 51 synthesis genes, while wCfeJ carries a CI-inducing toxin-antidote operon. Our analyses 52 of these genes highlight their mobility across the Wolbachia phylogeny and source to 53 other intracellular bacteria. Remarkably, the C. felis genome also carries two CI-like 54 antidote genes divergent from the wCfeJ antidote gene, indicating episodic RP in cat 55 fleas. Collectively, wCfeT and wCfeJ inform on the rampant dissemination of diverse 56 factors that mediate Wolbachia strategies for persisting in invertebrate host populations. 57 58 Key words 59Wolbachia, Ctenocephalides felis, cat flea, reproductive parasitism, mutualism, lateral 60 gene transfer, cytoplasmic incompatibility, biotin operon 61 killing have been characterized (24-29) and occur predominantly in the eukaryotic 87 association module (EAM) of Wolbachia prophage genomes (30). These genes 88 highlight the role of LGT in providing wolbachiae with factors facilitating mutualism or 89 RP, both of which are highly successful strategies for increasing infection frequency in 90 invertebrate host populations. 91Compared to reproductive parasites, Wolbachia mutualists appear to form more 92 stable, long-term relationships with their hosts, as supported by Wolbachia-host 93 codivergence in certain filarial nematodes (31) and Nomada bees (32). In contrast to 94 the stability of mutualists, relationships of reproductive parasites appear more 95 ephemeral. RP can be a strong mechanism to increase infection frequency, and CI 96 inducing strains can replace populations without infections (33, 34). Despite this, CI is 97 prone to neutralization through the evolution of host suppression (23, 35, 36) and 98 purifying selection on the host doesn't preserve CI (36). A weakened CI background 99 might be a ripe setting for invasions to begin. Whether invasion occurs at the level of 100 alternate wolbachiae, WO-phages, or CI operons themselves is an area of active 101 evolutionary research, but a clear result of this evolutionary complexity is that RP 102inducing Wolbachia phylogenies are discordant with those of their hosts (37-39). 103Horizontal transmissions, which can occur through direct host interactions, 104 environmental contacts (shared habitat, food sources, etc.) or predator/parasitoid 105 delivery, contribute to the discordance (40) and possibly drive episodic invasions and 106 replacements. 107Horizontal transmission is also evident from...

Identification of microbial taxa present in Ctenocephalides felis (cat flea) reveals widespread co-infection and associations with vector phylogeny

“…The high proportion of fleas with coinfecting Wolbachia ASVs is on par with previous evidence of Wolbachia co-infection in laboratory flea colonies. Dricoll et al proposed a selection for wCfeT over wCfeJ in nature, which agrees with our finding that ASV6 (100% wCfeT) was the dominant Wolbachia ASV and ASV383 (100% wCfeJ) was observed in only a single flea [26]. An important limitation of the present study was the low biomass of each C. felis sample, which likely allowed contaminant DNA from extraction kits and cross-contamination to dominate data [28].…”

“…The most abundant Wolbachia ASV (ASV6) aligned perfectly with wCfeT (accession ID NZ_CP051156.1). ASV383, the fourth most abundant Wolbachia ASV, aligned perfectly with wCfeJ (accession ID NZ_ CP051157.1) [26]. The full diversity of Wolbachia ASVs compared to wCfeT and wCfeJ is shown in Fig.…”

“…can exert a variety of effects on their hosts including manipulating host reproduction and altering pathogen transmission [23][24][25]. Research describing the C. felis-Wolbachia relationship, including the exchange of metabolites, epidemiology, and diversity of Wolbachia infecting C. felis, is in its infancy [26].…”

Extremely reduced supergroup F Wolbachia: transition to obligate insect symbionts

“…A particularly interesting case is presented by three pantothenate synthesis related genes (panB, panG and panC) found exclusively in wMeur1. The only Wolbachia homologues found by diamond blastx were genes from wClefT (NZ_CP051156), a strain infecting the cat ea Ctenocephalides felis (Driscoll et al 2020). The other closest relatives originated from phylogenetically distant bacteria, including several symbiotic forms.…”

Horizontal gene transfer-mediated bacterial strain variation affects host fitness

“…The most prominent may be the acquisition of vitamin B7 (biotin) synthesis by bed bugassociated Wolbachia [83]. Similarly, cat flea-associated Wolbachia seem to have gained the ability to produce biotin via HGT from closely related strains [84]. In ticks, pabA (and possibly pabB) required for the synthesis of folic acid, was acquired by a Coxiella like symbiont through horizontal gene transfer (HGT) from an Alphaproteobacterium [85] and is thought to affect tick fitness.…”