Wolbachiae are obligate intracellular bacteria that infect arthropods and certain nematodes. Usually maternally inherited, they may provision nutrients to (mutualism) or alter sexual biology of (reproductive parasitism) their invertebrate hosts. We report the assembly of closed genomes for two novel wolbachiae,wCfeT andwCfeJ, found co-infecting cat fleas (Ctenocephalides felis) of the Elward Laboratory colony (Soquel, CA, USA).wCfeT is basal to nearly all describedWolbachiasupergroups, whilewCfeJ is related to supergroups C, D and F. Both genomes contain laterally transferred genes that inform on the evolution ofWolbachiahost associations.wCfeT carries the Biotin synthesis Operon of Obligate intracellular Microbes (BOOM); our analyses reveal five independent acquisitions of BOOM across theWolbachiatree, indicating parallel evolution towards mutualism. Alternately,wCfeJ harbors a toxin-antidote operon analogous to thewPipcinABoperon recently characterized as an inducer of cytoplasmic incompatibility (CI) in flies.wCfeJcinBand three adjacent genes are collectively similar to large modular toxins encoded in CI-like operons of certainWolbachiastrains andRickettsiaspecies, signifying that CI toxins streamline by fission of large modular toxins. Remarkably, theC.felisgenome itself contains two CI-like antidote genes, divergent fromwCfeJcinA, revealing episodic reproductive parasitism in cat fleas and evidencing mobility of CI loci independent of WO-phage. Additional screening revealed predominant co-infection (wCfeT/wCfeJ) amongstC.feliscolonies, though fleas in wild populations mostly harborwCfeT alone. Collectively, genomes ofwCfeT,wCfeJ, and their cat flea host supply instances of lateral gene transfers that could drive transitions between parasitism and mutualism.

Rickettsia felis is an emerging pathogen of the transitional group of Rickettsia species and an important cause of febrile illness in Africa. Since the organism’s original discovery in the early 1990s, much research has been directed towards elucidating transmission mechanisms within the primary host and reservoir, the cat flea (Ctenocephalides felis). Several mechanisms for vertical and horizontal transmission within this vector have been thoroughly described, as well as transmission to other arthropod vectors, including other species of fleas. However, while a growing number of human cases of flea-borne spotted fever are being reported throughout the world, a definitive transmission mechanism from arthropod host to vertebrate host resulting in disease has not been found. Several possible mechanisms, including bite of infected arthropods and association with infectious arthropod feces, are currently being investigated.

The geographical overlap of multiple Rickettsia and tick species coincides with the molecular detection of a variety of rickettsial agents in what may be novel tick hosts. However, little is known concerning transmissibility of rickettsial species by various tick hosts. To examine the vertical transmission potential between select tick and rickettsial species, two sympatric species of ticks, Dermacentor variabilis and Amblyomma maculatum, were exposed to five different rickettsial species, including Rickettsia rickettsii, Rickettsia parkeri, Rickettsia montanensis, Rickettsia amblyommatis, or flea-borne Rickettsia felis. Fitness-related metrics including engorgement weight, egg production index, nutrient index, and egg hatch percentage were then assessed. Subsamples of egg clutches and unfed larvae, nymphs, and adults for each cohort were assessed for transovarial and transstadial transmission of rickettsiae by qPCR. Rickettsial exposure had a minimal fitness effect in D. variabilis and transovarial transmission was observed for all groups except R. rickettsii. In contrast, rickettsial exposure negatively influenced A. maculatum fitness and transovarial transmission of rickettsiae was demonstrated only for R. amblyommatis- and R. parkeri-exposed ticks. Sustained maintenance of rickettsiae via transstadial transmission was diminished from F1 larvae to F1 adults in both tick species. The findings of this study suggest transovarial transmission specificity may not be tick species dependent, and sustained vertical transmission is not common.

The Gram-negative obligate intracellular bacterium Rickettsia parkeri is an emerging tick-borne human pathogen. Recently, R. parkeri Sca2 and RickA have been implicated in adherence and actin-based motility in vertebrate host cell infection models; however, the rickettsia-derived factors essential to tick infection are unknown. Using R. parkeri mutants lacking functional Sca2 or RickA to compare actin polymerization, replication, and cell-to-cell spread in vitro , similar phenotypes in tick and mammalian cells were observed. Specifically, actin polymerization in cultured tick cells is controlled by the two separate proteins in a time-dependent manner. To assess the role of Sca2 and RickA in dissemination in the tick host, Rickettsia -free Amblyomma maculatum , the natural vector of R. parkeri , was exposed to wild-type, R. parkeri rickA :: tn , or R. parkeri sca2 :: tn bacteria, and individual tick tissues, including salivary glands, midguts, ovaries, and hemolymph, were analyzed at 12 h and after continued bloodmeal acquisition for 3 or 7 days postexposure. Initially, ticks exposed to wild-type R. parkeri had the highest rickettsial load across all organs; however, rickettsial loads decreased and wild-type rickettsiae were cleared from the ovaries at 7 days postexposure. In contrast, ticks exposed to R. parkeri rickA :: tn or R. parkeri sca2 :: tn had comparatively lower rickettsial loads, but bacteria persisted in all organs for 7 days. These data suggest that while RickA and Sca2 function in actin polymerization in tick cells, the absence of these proteins did not change dissemination patterns within the tick vector.

Vector-borne pathogens must overcome arthropod infection and escape barriers (e.g. midgut and salivary glands) during the extrinsic incubation period (EIP) before subsequent transmission to another host. This particular timespan is undetermined for the etiological agent of flea-borne spotted fever (Rickettsia felis). Artificial acquisition of R. felis by blood-feeding cat fleas revealed dissemination to the salivary glands after seven days; however, this length of time is inconsistent with co-feeding studies that produced infectious cat fleas within 24 h of infection. In the current study, we demonstrated that an alternative mechanism is responsible for the early-phase transmission that typifies flea-borne R. felis spread. Co-feeding transmission bioassays were constructed to assess temporal dynamics of R. felis amongst cat fleas, including exposure time to produce infectious fleas and association time to transmit infection to naïve fleas. Additional experiments examined the proportion of R. felis-exposed cat fleas with contaminated mouthparts, as well as the likelihood for cat fleas to release R. felis from their mouthparts following exposure to an infectious bloodmeal. The potential for mechanical transmission of R. felis by co-feeding cat fleas was further examined using fluorescent latex beads, as opposed to a live pathogen, which would not require a biological mechanism to achieve transmission. Analyses revealed that R. felis-infected cat fleas were infectious to naïve fleas less than 24 h after exposure to the pathogen, but showed no rickettsial dissemination to the salivary glands during this early-phase transmission. Additionally, the current study revealed that R. felis-infected cat fleas must co-feed with naïve fleas for more than 12 h in order for early-phase transmission to occur. Further evidence supported that contaminated flea mouthparts may be the source of the bacteria transmitted early, and demonstrated that R. felis is released from the mouthparts during brief probing events. Moreover, the use of fluorescent latex beads supports the notion that early-phase transmission of R. felis is a mechanical mechanism. Determination of the transmission mechanisms utilized by R. felis is essential to fully understand the vulnerability of susceptible vertebrate hosts, including humans, to this pathogen.