Unpacking the Intricacies of Rickettsia-Vector Interactions

Abstract

Although Rickettsia species are molecularly detected among a wide range of arthropods, vector competence becomes an imperative aspect of understanding the eco-epidemiology of these vector-borne diseases. The synergy between vector homeostasis and rickettsial invasion, replication, and release initiated within hours (insects) and days (ticks) permits successful transmission of rickettsiae. Uncovering the molecular interplay between rickettsiae and their vectors necessitates examining the multi-faceted nature of rickettsial virulence and vector infection tolerance. Here, we highlight the biological differences between tick- and insect-borne rickettsiae and the factors facilitating the incidence of rickettsioses. Untangling the complex relationship between rickettsial genetics, vector biology, and microbial interactions is crucial in understanding the intricate association between rickettsiae and their vectors.

Rickettsial Diversity and Vector Transmission

With historical significance and recognized resurgence and emergence, vector-borne rickettsial diseases affecting human health are of critical importance worldwide. In the United States, this growing trend is evident in a recent report of notifiable diseases with cases of tick-borne spotted fever rickettsiosis increasing by 23% from 2016-2018 ⁱ and the recent re-emergence of flea-borne rickettsiosis within endemic areas in the United States, including California and Texas [1]. Likewise, the reappearance of localized outbreaks of louse-borne epidemic typhus repeatedly occurs in displaced populations worldwide, where humans remain a natural reservoir host (see Glossary) [2]. Rickettsial diseases are caused by obligate intracellular bacteria transmitted by various hematophagous arthropods, including ticks, fleas, lice, and mites (Box 1), through infectious salivary secretions or feces. Thus, the determinants governing rickettsial biology and ecology are due to their inextricable link with the distribution and behavior of their corresponding vectors. Failure to understand the molecular factors fostering this dynamic life cycle (traversing between vectors and vertebrate hosts) leaves a significant gap in controlling the spread of these pathogens. Although specific virulence determinants of Rickettsia (Alphaproteobacteria: Rickettsiales: Rickettsiaceae:) remain elusive, several elements have been proposed to play an essential role in infection of both vector and vertebrate hosts, such as a reduction in genome size [3], expression of outer membrane proteins involved in adhesion or invasion [4, 5], metabolic reprogramming [6, 7], or rickettsial burden [8] (Figure 1). Understanding these factors is paramount, as minute genetic differences can be attributed to significant alterations in virulence [5, 9]. Fortunately, the diversity within the Rickettsia genus provides the opportunity to draw comparisons between those species with known pathogenicity in vertebrates and those that play symbiotic roles within their vector hosts. Appreciation of the factors contributing to vector competence, including the molecular basis of rickettsial virulence, arthropod biology, vector-host interface, and microbial interactions, serve as the foundation for examining the ecology of tick- and insect-borne rickettsioses.

Box 1. Mite transmission.

Mites are known vectors of clinically relevant human pathogens, such as Rickettsia akari (Rickettsialpox) and Orientia tsutsugamushi (scrub typhus), formerly referred to as Rickettsia tsutsugamushi. The house mouse (Liponyssoides sanguineus) and chigger Thrombiculid mite (Leptotrombidium spp.) are principal vectors of R. akari and O. tsutsugamushi, respectively. These mites have low host specificity but are commonly found on small mammals where zoonotic spillover to humans occurs in the absence of a preferred host [96]. Mites undergo a complicated life cycle with 4-7 stages, including egg, 6-legged larval stages, and 8-legged nymphal and adult stages [97]. Like tick-borne rickettsiae, R. akari undergoes both transovarial and transstadial transmission, suggesting L. sanguieus can act as a biological reservoir for the pathogen [98]. However, larval stages of Dermanyssid mites do not consume a blood meal; thus, they do not contribute to horizontal transmission [99]. Conversely, Thrombiculid mites do not partake in a blood meal but instead consume host lymphatic fluid and decomposed tissues by partially digesting host tissues with salivary secretions [97]. Moreover, larval Thrombiculid mites (chiggers) are the only parasitic stage for vertebrate hosts; thus, pathogen acquisition and horizontal transmission only occur at this developmental phase. Vertical transmission of O. tsutsugamushi by chiggers can be as high as 100%, with no adverse effects on vector viability or fecundity [97]. The biological differences between mite species present a unique co-evolution between mite-borne pathogens and their vectors to ensure successful maintenance in nature. With limited understanding of mite-borne transmission of R. akari and the increasing incidence of scrub typhus around the world [100], the opportunity to compare and contrast the unique transmission biology can provide further insight into the epidemiology of these mite-borne pathogens.

Figure 1. Attributes of rickettsiae guiding the interaction within arthropod vectors.

Upon early infection within their vector hosts, rickettsiae must modulate the arthropod immune response to facilitate survival, cellular invasion, and replication. Due to their intracellular lifestyle, all rickettsiae must sequester host metabolites to ensure replication. Distinguishing factors proposed that enhance virulence among rickettsial species include genome reduction or gene regulation [3]; the expression of Omps involved in adhesion, invasion, or motility [4, 5]; metabolic reprogramming [6, 7]; and increased bacterial loads within vector tissues, such as salivary glands [8]. Red depicts factors indicative of pathogens, and green represents attributes of endosymbionts. Pathogenic Rickettsia spp. tend to favor horizontal transmission due to potential detrimental effects on fecundity or fitness of their arthropod hosts; therefore, decreasing the efficiency of vertical transmission [11, 12]. However, increasing reports have incriminated rickettsial endosymbionts as causes of disease in vertebrates inferring their ability also to be horizontally transmitted [19]. A defining characteristic of endosymbionts is their high-efficiency rates of vertical transmission within vector populations [11], which is facilitated by their ability to provide nutritional symbiosis to their vector hosts [9]. Additionally, restrictions such as truncated Omps are thought to play a role in tissue dissemination, limiting rickettsial spread to salivary glands [4]. Confounding the virulence spectrum is the presence of plasmids among Rickettsia species, while common among most rickettsial endosymbionts, species known to be pathogenic to humans also possess these genetic elements [3, 44]. Thus, their function remains unknown. Abbreviations: Omps, outer membrane proteins.

The genus Rickettsia contains 31 recognized species ⁱⁱ and several candidate species or uncharacterized strains with known variation in pathogenicity and arthropod hosts, further complicating the eco-epidemiology of these emerging and re-emerging diseases. One of the unique aspects of rickettsial biology is the variation in transmission mechanisms. While several Rickettsia species are strictly maintained vertically within arthropod populations, others exhibit mixed (horizontal and vertical) transmission. It is appreciated that frequent horizontal (infectious) transmission favors virulent rickettsiae, whereas vertical (inherited) transmission favors the evolution of benign and mutualistic associations [10]. For example, some virulent Rickettsia spp. are detrimental to vector fitness or fecundity, contributing to their reduced prevalence rates among arthropod populations and reliance on horizontal transmission events to persist in nature [11-13] (Box 2). In contrast, rickettsial endosymbionts tend to have high prevalence rates in vector populations due to their symbiotic relationships. Surveillance data implicates rickettsial endosymbionts as major contributors to tick microbiomes [14, 15], suggesting a role for nutritional symbiosis [16]. Moreover, recent reports have incriminated what were considered rickettsial endosymbionts as potential or known causes of human disease [17-19], clouding the distinction between rickettsial endosymbionts and pathogens.

Box 2. Rickettsial pathogenicity and vector competence.

The worldwide presence of rickettsiae mirrors genotypic differences among Rickettsia species from different vectors, vertebrate hosts, and geographic regions [12, 22, 101, 102]. With the known lethal effects of R. prowazekii on louse viability, tick fitness has also been examined in relation to pathogenic tick-borne rickettsiae. Historically understood, R. rickettsii maintains low vertical transmission efficiency within ticks, which is correlated to the virulence of the strain used under laboratory conditions [103, 104]. It is thought that the inefficiency of R. rickettsii to be vertically maintained is due to the inability of the pathogen to sufficiently colonize the ovaries of adult female ticks [103]. More recent experimental infections of Amblyomma tick spp. with globally dispersed R. rickettsii isolates depict a unique, intricate relationship between rickettsiae and vector competence [104, 105]. Ticks infected with R. rickettsii isolates circulating within the same natural geographical range have enhanced acquisition and transmission when compared to those ticks infected with an isolate circulating outside their natural range. Yet, a reduction in fecundity was observed, regardless of sympatric relationships. Additionally, R. conorii infection of its natural tick vector, Rhipicephalus sanguineus, also proves to be detrimental to tick viability. Although R. conorii strain Malish 7 is of African origin, it is detrimental to tick fitness for both North American and Mediterranean R. sanguineus ticks compared to that of a Mediterranean isolate [12]. The adverse effects of R. conorii strain infection on tick survival presents a contrasting scenario whereby these two R. conorii strains may utilize alternative routes of transmission to maintain infections in nature as opposed to a relationship governed by geographic overlap as observed for R. rickettsii. Due to some rickettsial pathogens causing adverse effects on their tick hosts, the necessity of horizontal transmission events and essential amplifying vertebrate hosts is central to the maintenance of virulent Rickettsia species in nature. A thorough understanding of the impact of rickettsial infection on tick vector fitness is required, especially when considering the recent introduction of new tick species and the emergence of rickettsial infections in many parts of the world. Of interest is the capacity of an invasive tick in the United States, Haemaphysalis longicornis, to transmit rickettsiae. Both vertical and horizontal transmission of R. rickettsii can occur under laboratory conditions, but its role in the epidemiology of tick-borne rickettsioses remains to be elucidated [106].

The complexity of rickettsiae-vector interrelationships is further perpetuated by intra-specific variation of geographically distinct rickettsial strains in both vertebrate and vector hosts [12, 20-23]. For example, Rickettsia felis genetic variants are both spatially and biologically distinct, with one an obligate symbiont of the parthenogenic booklouse and others transmitted by cat fleas [24]. Cat fleas can acquire and horizontally transmit either genotype, regardless of host derivation, illustrating the non-exclusive nature of cat fleas to transmit multiple rickettsial genotypes [25]. In contrast, Amblyomma maculatum ticks appear to be more susceptible to spotted fever group (SFG) rickettsiae infection and vertical transmission than Dermacentor variabilis ticks during experimental exposure [26]. Therefore, it is likely that individual rickettsial species can infect many arthropods, with successful biological transmission being dependent on undefined rickettsiae- and vector-specific factors.

The Biology of Rickettsial Infection in Arthropods

Rickettsiae can be acquired through feeding on a bacteremic host. Alternative acquisition routes have also been described, including co-feeding with an infected arthropod on a non-rickettsemic host or transovarial transmission. Due to the host cell requirement for rickettsiae to replicate, a critical initial step in rickettsial pathogenesis is the bacterial recognition of and attachment to target cells. Nevertheless, the study of Rickettsiology has been hampered by the lack of genetic tools to interrogate virulence determinants. While pioneering studies have laid the groundwork [27], only more recently has the process been streamlined to generate both random and site-directed genetic mutants [28-30]. The availability of these mutants has ushered in a new appreciation of the biology of rickettsiae in vertebrate hosts, capitalizing on several in vitro and in vivo models of infection and disease [29, 31]. Although our understanding of rickettsial infection in vertebrate cells is accumulating [32], the molecular requirement for rickettsiae to infect and disseminate through arthropods is generally lacking. Factors essential for typical rickettsial infection in vertebrate host cells [33] have been shown to play confounding roles within the vector. Some tick-borne rickettsiae utilize host-derived molecules to assemble actin tails facilitating movement to propel themselves from one cell to the next. Although ticks contain the host cell machinery to promote rickettsial-induced actin-based motility (ABM), the process is not essential for disseminated tick infections and may even counter persistence within tick ovaries [34].

The ability of rickettsiae to invade, replicate, and disseminate within vector hosts is inherently coupled with the physiology of arthropod blood-feeding and digestion. Insects, such as fleas and lice, partake in frequent, intermittent blood meals, owing to rapid blood meal digestion [35]. In contrast, ixodid ticks engorge themselves during one prolonged feeding event at each hematophagous life stage. The distinct feeding habits of these arthropods contribute to variation at the vector-pathogen interface. Specifically, in the arthropod midgut, rickettsiae are exposed to a milieu of molecules, ranging from antimicrobial peptides (AMPs) and reactive oxygen species (ROS) to nutrients. While blood provides the arthropod with protein-rich metabolites necessary for development and egg production, the process of breaking down hemoglobin releases dangerous AMPs and toxic heme into the midgut lumen, where newly acquired rickettsiae can be targeted [36]. For arthropods, the release of toxins during blood meal digestion can be quite dangerous. Therefore, several species form a peritrophic matrix (PM) around the blood meal to shield the midgut epithelial cells from ingested microbes or digested particles [37, 38]. However, this protective sheath appears to be absent in fleas and lice [39] and several tick species, except Ixodes and some Haemaphysalis spp. [38]. However, the facilitative or refractory role of the PM during rickettsial infection remains undefined. Unique to ticks, sequestering of toxic heme occurs by digesting hemoglobin within hemosomes, thereby inadvertently harboring rickettsiae from some of these lytic compounds. However, mechanisms of survival by rickettsiae within the midgut microenvironment remain unknown.

Rickettsiae must evade arthropod defense mechanisms at the site of ingestion (midgut), route of travel (hemolymph), and proximal tissues (e.g., salivary glands, ovary) to achieve disseminated infection and subsequent transmission to a new host. Hematophagous arthropods mount innate defenses in response to blood-feeding and microbial insult via two major components: cellular defenses (hemocytes) and humoral responses (secretion of AMPs). A major contribution of AMPs occurs in the hemolymph where, interestingly, rickettsiae have been observed within 12 hours of exposure [34]. However, further investigation is needed to understand the physical state and active mechanisms utilized by rickettsiae during arthropod infection. While several mechanisms of innate immunity in arthropod vectors have been identified [40], their exact role during species-specific infection remains unknown. Differential activity of AMPs, when exposed to pathogens versus endosymbionts, presents a specific recognition in the tick background [41, 42]. Still undetermined is the specificity of rickettsiae-vector relationships guiding the balance between inhibiting infection and widespread dissemination in the vector. However, recently compiled arthropod genomes have armed the field with the capacity to utilize techniques, such as RNA interference (RNAi) and CRISPR-Cas9, in arthropods to enhance our understanding of factors driving vector competence [40, 43].

Tick-borne Rickettsiae

Classification of the Rickettsia genus is based on genetic and biological characteristics [44]. Tick-borne, SFG rickettsiae consist of species ranging from those that cause severe and often fatal human diseases, such as Rocky Mountain spotted fever, to strict tick endosymbionts. To ensure persistence within tick populations, tick-borne rickettsiae rely on both transovarial and transstadial transmission. If imbibed in a blood meal, it is presumed that rickettsiae traverse through the midgut epithelium into the hemocoel and subsequently infect other tissues, such as the ovary or salivary glands (Figure 2, Key Figure). Infection via cells lining the tracheal system has also been proposed for rickettsial dissemination in ticks, as viruses in other arthropods readily utilize this mechanism. The tracheal system remains relatively intact during the molting periods, in contrast to the salivary glands, which undergo significant developmental changes [45]. The precise mechanisms by which tick-borne rickettsiae achieve multi-tissue infections require further examination.

Figure 2, Key Figure. Transmission mechanisms of rickettsiae.

After arthropods imbibe an infectious bloodmeal, rickettsiae encounter the MG of ticks, lice, or fleas where recognition causes the secretion of various soluble effectors, such as AMPs (A, G), ROS (A, D, G), serine proteases (G), and serpins (G) into the MG lumen. As rickettsiae attach to unknown receptors and invade MG epithelial cells, interactions with resident endosymbionts occur (A, D, H). The rapid digestion of host cells in insect vectors such as lice and fleas prompt efficient attachment to MG epithelial cells and internalization of rickettsiae to avoid destruction by proteolytic compounds (D, G). For tick-borne rickettsiae, it is presumed that rickettsiae are internalized by receptor-mediated endocytosis of MG epithelial cells initiated by the digestive process of Hb (B). Tick-borne rickettsiae spread cell-to-cell, ultimately traversing to the H, where they gain access to both the SG and OV for transmission (C). As insect-borne rickettsiae replicate, host cell lysis occurs, releasing rickettsiae back into the MG lumen enabling transmission to vertebrate hosts through the inoculation of infectious feces in abrasions of the skin (E, I). Although increased MG epithelial permeability causes mortality of lice vectors (F), flea-borne rickettsiae can navigate to the H, disseminating to SG and OV for transmission (I). Green and orange arrows represent arthropod/vertical and vertebrate/horizontal transmission, respectively. Abbreviations: AMPs, antimicrobial peptides; H, hemocoel; Hb, hemoglobin; MG, midgut; OV, ovaries; RBCs, red blood cells; ROS, reactive oxygen species; serpins, serine protease inhibitors; SG, salivary gland.

As rickettsiae enter the tick midgut lumen during blood meal acquisition, microbial proliferation is favorable [38, 46]. Therefore, ticks must develop strong immune defenses to protect themselves against the invasion of pathogens, such as the secretion of AMPs by the epithelial cells via stimulation of the immune deficiency (IMD) or Toll pathway [47-50]. Specifically, a Kunitz protease inhibitor directly interacts with rickettsiae in the midgut lumen before the invasion of epithelial cells [51]. Experimental exposure of Amblyomma ticks to Rickettsia rickettsii infection induces midgut differential expression of genes involved in metabolism, protein modification, and secreted proteins compared to uninfected controls [47]. Intriguingly, these transcripts are also differentially expressed between two R. rickettsii-infected Amblyomma tick species, potentially contributing to their differences in vector competence. The differential modulation of the tick immune system by rickettsiae is under investigation, providing additional insight on the factors influencing vector competence between arthropod species.

Adult ticks acquire a blood meal over a prolonged period (7-10 days) where digestion begins slowly (3-6 days after the initiation of feeding) and may last several months [38]. Hemosomes limit exposure to deleterious enzymes apart from hemocidins and ROS, which are still released into the midgut lumen [38, 47]. While ROS possesses anti-microbial properties, it also promotes apoptosis of tick cells [52]. Therefore, ticks reduce oxidative stress by isolating heme and producing antioxidant enzymes and several selenoproteins. Antioxidant enzymes are differentially expressed during different blood-feeding stages, tissues, and when exposed to various Rickettsia species, facilitating rickettsial vertical transmission or tick colonization [47, 53]. To escape ROS damage, rickettsiae are thought to exploit the engulfment of hemoglobin by epithelial cells as a mechanism of entry [54]. Moreover, hemosomes are slow to fuse with the lysosome, thus allowing ample time for rickettsiae to escape from the endosome and begin replication within the cytoplasm [46, 54]. Manipulating the balance between activation of an innate defense system during blood meal acquisition and mitigating microbial infections provides new opportunities to control tick-borne rickettsial pathogens.

Once tick-borne rickettsiae traverse the midgut cells and enter the hemocoel, they must cope with several adverse physiological mechanisms to survive. Hemocytes utilize lectin-mediated recognition of pathogen-associated molecular patterns produced by microbes to trigger the release of AMPs, recruit additional phagocytic cells, and directly phagocytize invading microbes. AMPs, such as serine proteases, present in small cytoplasmic granules of some hemocytes can facilitate the degradation and destruction of pathogens incorporated by cells or free in the hemolymph [38, 50]. The presence of rickettsiae in tick hemolymph [34, 45, 55] suggests that the bacteria have developed countermeasures to the basic tick anti-microbial response, yet the evasion mechanisms of rickettsiae remain uncharacterized.

Upon dissemination to the salivary glands, rickettsiae must evade salivary gland secreted factors, such as AMPs, proteases, ML-domain proteins, histidine-rich proteins, and peptidoglycan recognition proteins (PGRP) [48, 56]. The kinetics of rickettsial salivary gland colonization leading to successful transmission, explicitly examining the specific rickettsiae- and tick-dependent mechanisms, require further elucidation. Currently, the assignment of rickettsial virulence is based on in vitro assays or needle inoculated animals. Recognizing that in nature, ticks deliver rickettsiae to vertebrate hosts in a protein-rich saliva vehicle, uncoupling the vector from the inoculation event fails to capture the transition of rickettsiae at the tick-host interface. Saliva-assisted transmission is thought to occur via the secretion of pharmacologically active molecules produced by the tick and has been studied in tick-borne pathogen transmission models [57, 58] (Box 3). The extent to which rickettsiae manipulate the production of tick-derived factors essential for successful feeding and subsequent transmission to a vertebrate host needs to be examined.

Box 3. Tick salivary secretions facilitate pathogen transmission.

Tick saliva not only provides a matrix for pathogen transmission but also is comprised of an assortment of molecules regulating the host immune response that facilitates blood meal acquisition. At the cutaneous interface, saliva-assisted transmission (SAT) has been documented for several human pathogens in ixodid ticks, such as Borrelia species, Arboviruses, Anaplasma phagocytophilum, and Bartonella henselae [57]. The role of Ixodes tick SAT of Borrelia burgdorferi sensu lato (s.l.) is well known. Specifically, the salivary gland protein Salp15 is one of the first and well-characterized moderators of SAT in ticks. By directly binding to the outer surface protein OspC, Salp15 protects B. burgdorferi s.l. from antibody- and complement-mediating killing at the site of tick-feeding. Confirmed by RNAi silencing and mouse immunizations with Salp15, this lipoprotein plays a pivotal role in the transmission of Borrelia to a vertebrate host. Other molecules implicated as facilitators of SAT in Ixodes ticks include Salp20, Salp25D, tick salivary lectin pathway inhibitor, Saliostatin L2, tick histamine release factor, and B cell inhibitory protein all functioning as mediators of host immunological responses to protect pathogen disposition [57, 58]. Collectively, Sialostatin L2 interferes with mouse dendritic cell responses during tick-borne encephalitis virus and Borrelia infection while inhibiting inflammasome formation in mice during A. phagocytophilum infection portraying a multi-functional role of tick salivary antigens. Interestingly, SAT by Ixodes ricinus ticks of a common flea-borne pathogen, B. henselae, implicates an interaction with a serine protease inhibitor (IrSPI), but its precise mechanism is undefined. While tick-borne rickettsiae are known to influence salivary gland biology [56, 107] the specific interactions remain elusive.

It was traditionally appreciated that rickettsial endosymbionts were restricted to tick reproductive tissues due to the lack of functional genes found in virulent rickettsiae [4, 59]. The recent identification of rickettsial endosymbionts in tick salivary glands suggests transmission potential [8, 45, 60, 61]. Interestingly, while both pathogenic and endosymbiont tick-borne rickettsiae disseminate to the salivary glands of infected ticks and are horizontally transmitted, pathogenic rickettsiae are detected earlier at higher loads in vertebrate hosts. It is proposed that the increased virulence of rickettsiae perpetuated a higher rickettsial burden within the tick, contributing to disease [8]. Understanding the Rickettsia species-specific influence associated with the physiological changes in infected salivary glands could provide potential targets in limiting transmission to vertebrate hosts.

Necessary for vertical maintenance of rickettsiae, the mechanisms of ovarian infection have not been fully elucidated. It is thought that oocytes engulf rickettsiae during an active process of endocytosis induced by the uptake of vitellogenin transported through the hemocoel [46]. The expression of several AMPs and other proteins involved in metabolism and oxidative stress are upregulated in tick ovaries in response to rickettsial infection [62, 63]. Yet, bacteria-specific receptors and other biological variables that govern the tick/rickettsiae infection process are undefined.

With three post-embryonic (subadult and adult) life cycle stages requiring a blood meal for ixodid ticks, microbial management of resources is necessary as the vector undergoes physiological and metabolic changes during this quiescent period between feeding events [38]. For tick-borne rickettsiae, this period of inactivity requires long-term storage of metabolites and escape from the tick's protective immune response. As a tick initiates feeding on a new host, changes in temperature and physiology were once considered requirements to confer rickettsial infectivity [64, 65]. Challenging this historical understanding, it appears that tick-borne rickettsiae reside within the salivary duct lumen and are primed for entry into the host within 30 minutes post-attachment [55, 66]. Fully plotting the acquisition, dissemination, and survival strategies of rickettsiae in ticks during horizontal and vertical transmission events is warranted. Providing details on the physiology and metabolic activity of rickettsiae as they alternate between vector and vertebrate hosts will offer insight into preferred target cells and tissues utilized during vector infection.

Insect-borne Rickettsiae

Another group of Rickettsia species, the typhus group (TG), includes pathogenic Rickettsia typhi and Rickettsia prowazekii, associated with fleas and lice, respectively. Possessing shared genetic characteristics between SFG and TG while maintaining distinguishable biological features, the transitional group (TRG) has been more recently classified [44]. The TRG contains species with varying pathogenicity, such as R. felis, that are associated with diverse arthropod hosts (e.g., fleas, lice, mites, and ticks). The versatile transmission routes of insect-borne rickettsiae include vertical and horizontal (salivary secretions and infectious feces). After ingestion through an infectious blood meal, rickettsiae invade the midgut epithelium, replicate, and are subsequently released back into the midgut lumen, where they are excreted in fecal matter (Figure 2). Insect-borne rickettsiae do not employ ABM to spread from cell-to-cell and instead are expelled back into the extracellular space through direct host cell lysis [35]. Constant peristalsis of the midgut enables prompt excretion through fecal matter where rickettsiae can remain infectious for several months [67]. Although long recognized as an avenue of transmission, the biological attributes of rickettsiae released at the time of defecation are unknown and require further attention to fully understand this route of exposure to a vertebrate host.

Rickettsial infection can have an adverse fitness effect on the vector. In the louse, R. prowazekii causes mortality six days following an infectious blood meal [13]. Rapid midgut cell lysis increases epithelium permeability, triggering midgut lumen contents to leak into the hemocoel (Figure 2). The louse becomes red due to the leakage of blood into its translucent body cavity. Humans become infected by scratching viable rickettsiae from infected lice feces or crushed louse bodies into the insect bite site or host mucosal membranes [2, 68]. Due to the detrimental effect on louse viability, it is appreciated that R. prowazekii is not vertically maintained and requires a vertebrate reservoir for transmission in nature [2].

In contrast to R. prowazekii in lice, flea-borne rickettsiae do not influence flea fitness. R. felis can be maintained vertically in flea populations for up to 12 generations without an additional infectious blood source [69]. Although vertical transmission of flea-borne rickettsiae [69, 70] is highly variable in laboratory flea cohorts [71], constitutively infected flea colonies have been established from wild flea populations indicating vertical transmission occurs in nature. Both R. felis and R. typhi have been detected in various flea tissues (e.g., hemocoel, ovaries, salivary glands, hindgut, fat body), and the necessity of disseminated infections in the transmission of flea-borne rickettsiae is under investigation.

Fleas and lice consume frequent blood meals daily with rapid turnover (within the first 6 hours of feeding) of midgut contents, creating a hostile environment for microbes [39]. Specifically, fleas are armed with a proventriculus at the foregut entrance to physically disrupt host cells at the onset of feeding. Insect digestive enzymes are free within the midgut lumen, producing hazardous by-products unfavorable for ingested rickettsiae [72]. Induced by blood-feeding alone, increased transcription of serine proteases, such as trypsin and chymotrypsin, serine protease inhibitors, PGRP, and defensin, occurs [73]. This not only provides critical digestive enzymes but also presents an early defense mechanism against invading pathogens. Thus, rickettsiae imbibed during a blood meal must invade the midgut epithelium quickly; however, specific adhesion/invasion receptors in the insect vector remain to be identified. During experimental R. typhi infection of cat fleas, trypsin- and chymotrypsin-like molecules and putative GTPases, such as C. felis signal recognition particle and rab5, display differential regulation in the midgut compared to that of blood-feeding alone [74]. Although the anti-microbial response to insect-borne rickettsiae is limited, RNAi knockdown of IMD transcripts in the midguts of cat fleas enhanced R. typhi burden [75]. Interestingly, defensins (secreted AMPs) are not upregulated in the cat flea midgut in response to R. typhi infection [74] but are upregulated during rickettsial infection in the midgut of ticks [47, 63]. Alternatively, body lice have a simplified immune system that lacks a functional IMD recognition pathway, subsequently increasing their susceptibility to infection [76]. The distinct expression patterns observed in insects compared to that in ticks during rickettsial challenge provide an exciting platform to analyze differences in the ability of insect- and tick-borne rickettsiae to modulate their vectors.

Although the midgut remains the focal site of rickettsial colonization for flea and louse infections, the hemocoel is thought to be the route utilized by flea-borne rickettsiae to achieve multi-tissue infection. Similar to tick-borne rickettsiae [45], R. felis has been observed in the cat flea tracheal system, providing an alternative route of dissemination within the insect vector [77]. While the exact role of hemocytes during flea infection is less understood, the process of melanization has been proposed, as lectin-like molecules have been described during transcriptomic analyses [78]. Due to efficient fecal transmission, limited attention has been given to the transmission of insect-borne rickettsiae through salivary secretions. However, a sialotranscriptome of the cat flea revealed several flea molecules that may be influenced by rickettsial infection, including AMPs and targets of the Toll or IMD pathways [79]. Furthermore, the synergistic or regulatory activities of flea salivary gland secreted molecules during rickettsial infection and transmission to vertebrate hosts are in the early stages of characterization [80]. With both R. typhi and R. felis detected within salivary glands of infected fleas, further investigation is warranted to determine the impact of flea saliva on transmission.

Microbial Interactions and Vector Biology

In addition to vectors' chitinous exoskeleton, resident microflora can also defend against invading microorganisms. The role of endosymbionts in the transmission of vector-borne pathogens has gained increased attention due to their ability to alter an arthropod's vectorial capacity [40, 43, 81]. The influence of the microbiome in altering rickettsial vector immunity and metabolism is being assessed [53]. For example, tick endosymbionts in the genera Coxiella, Rickettsia, and Francisella, which are the most abundant microbes detected in tick populations [15, 16], provide nutrients deficient in the blood meal [82, 83]. Although not experimentally tested, the rickettsial endosymbiont plasmid (pREIS2) encodes for a biotin operon, which has been identified in wolbachiae mutualists, suggesting nutritional symbiosis [14]. In return, ticks have co-evolved measures to preserve the integrity of their microbiota, such as modulating the production of antioxidant molecules [53]. The intricate role of endosymbiont bacteria in maintaining tick homeostasis and their potential influence on vector competence requires further analysis.

Fleas supplement microbial diversity via active larval feeding on environmental debris. The strict hematophagous diet of adults lacks essential vitamins or cofactors necessary for proper development and requires endosymbiotic bacteria, including Wolbachia spp. [84]. Like the common louse endosymbiont (Candidatus Riesia), wolbachiae are known to provide essential biosynthetic pathways lacking in the blood meal, such as B vitamins, to its insect host [85, 86]. Furthermore, Wolbachia spp. manipulate insect reproductive fitness, potentially influencing disease ecology [81, 86]. While the interaction between competitive, vertically transmitted wolbachiae and rickettsiae has recently been examined in parasitoid wasps [87], the interaction remains understudied in fleas. Infection with R. felis has shown to decrease cat flea microbial richness [88], emphasizing the potential impact of vertically maintained rickettsiae on the robustness of the flea microbiota. Exploiting the microbiome contribution to vector competence may serve as a new approach to control vector-borne rickettsial diseases.

Further obscuring the current epidemiology of rickettsioses is the presence of multiple rickettsial species/strains in sympatric geographic areas [89, 90]. As arthropods consume multiple blood meals, there is an increased potential to acquire more than one organism, which influences the transmission biology of pathogens. For example, co-infections with rickettsiae can alter vertical transmission in ticks [91, 92]. Furthermore, Rickettsia amblyommatis (once considered a strict endosymbiont) undergoes disseminated infection in Amblyomma ticks [11] and is transmissible through a tick bite to vertebrate hosts [11, 89]. During dual infections, R. amblyommatis, provisionally designated as "Rickettsia amblyommii" [93], influences vector competence for both R. parkeri [61] and R. rickettsii [89]; however, the significance of this relationship must be further explored to determine the eco-epidemiological impact in nature. Comparable to tick-borne epidemiology, the recent discovery of R. felis genotypic variants, collectively referred to as R. felis-like organisms (RFLOs), in various arthropods throughout the world has complicated the understanding of flea-borne rickettsioses [90]. Although once presumed as strict endosymbionts, RFLOs have been molecularly detected among vertebrate hosts [94]. Furthermore, cat fleas constitutively infected with R. felis experimentally acquire R. typhi at a lower frequency after subsequent exposure compared to uninfected cat fleas [95]. Mechanisms such as cellular changes within oocytes, priming of the vector immune response, or defensive symbiosis have been proposed as components that may alter vector susceptibility to superinfections [92]. The components governing rickettsial co-infections and the effect on bacterial acquisition, maintenance, and transmission remain undefined.

Concluding Remarks

The diversity of Rickettsia genomes and transmitting vectors provide a rich substrate to interrogate host-pathogen interactions (see Outstanding Questions). Untangling the complex events in which rickettsiae can adapt to the dynamic shift between vector and vertebrate hosts will enable a better understanding of the molecular drivers associated with transmission. Limiting rickettsial disease management is the scant knowledge of the molecular and biological factors conveying rickettsial virulence and the vector-derived determinants of transmission. The unique feeding behaviors of pan-Arthropoda vectors of rickettsiae require a detailed examination of biologically relevant transmission models. Ticks are responsive to rickettsial infection and the extent to which Rickettsia spp. manipulate the vector for successful transmission needs to be identified. Providing multiple routes of transmission, insect vectors offer a unique opportunity to examine rickettsiae in different physiological states. Central to all arthropod vectors of rickettsiae is the underlying microbial influence on rickettsial transmission. Thus, by combining rickettsial genomics, studies of vector ecology and biology, and biologically relevant transmission models, molecular determinants facilitating transmission of obligate intracellular pathogens will be elucidated.

Outstanding Questions.

What are the molecular drivers of rickettsial prevalence among vector populations? Can discrete genetic differences between strains that confer vector competence, virulence, or geographic distribution be identified?

What are the biological and genetic factors defining rickettsial endosymbionts and pathogens? Are gene expression patterns different within different vector tissues?

How do rickettsiae manipulate their vector hosts to enable transmission? Do rickettsiae alter arthropod salivary secretions to facilitate transmission in a species-specific manner?

Can the biological properties of insect-borne rickettsiae shed in the feces provide a platform to discover unique genetic attributes of environmental persistence?

What are the ecological and epidemiological implications of vector microbiota and rickettsial co-infections on the emergence of rickettsioses?

Highlights.

The incidence of rickettsioses is constricted by the geographic range, host preference, and feeding habits of their vector hosts.

Ranging from strict vector endosymbionts to severe human pathogens, Rickettsia evolved several mechanisms fostering their dynamic life cycles (navigating between vector and vertebrate hosts).

A distinct vector response to Rickettsia emphasizes an intricate Rickettsia-vector relationship, but our understanding of these associations remains limited.

Vector competence must be an interplay between rickettsial genetics, vector biology and feeding habits, and sympatric microbial interactions.

Glossary

Antimicrobial peptides (AMPs)

molecules that are secreted as an innate immune response to an infection via IMD or Toll pathways. AMPs directly act against microbial agents by causing membrane disruption, inhibition of membrane protein synthesis, metabolism interference, or direct lysis. These molecules can include defensins, varisins, lysozymes, lectins, and protease inhibitors

Antioxidant enzymes

enzymes produced to decrease the effects of ROS, including catalase, superoxide dismutases, and glutathione-S-transferase

Bacteremic

refers to bacteria circulating within the blood of a vertebrate host that arthropods could subsequently acquire during the imbibement of a blood meal

Biological transmission

facilitating the replication and maintenance of a microbe, as opposed to mechanical transmission, or the movement of microbes by contamination

Co-feeding

refers to the simultaneous feeding of multiple vectors on a single host where organisms are transferred from an infected vector to a naïve (uninfected) vector, often in the absence of a systemic infection. This phenomenon contributes to pathogen spillover when multiple arthropod species feed on the same vertebrate host in nature

Constitutively infected

refers to arthropods naturally retaining an infection throughout their life stages (e.g., the organism is vertically maintained throughout generations)

Defensive symbiosis

the ability of commensal microbes to protect the host from subsequent microbe invasion by occupying niches or secreting antimicrobial factors

Eco-epidemiology

a term used to describe both the ecological and disease aspects that shape the spread of zoonotic pathogens

Endosymbionts

vertically transmitted intracellular organisms that often portray an intimate relationship with their host, affecting fitness, development, reproduction, or immunity

Fat body

a lipid-rich tissue distributed throughout the hemocoel of arthropods that is a significant source of secreted AMPs, analogous to the liver in vertebrates

Fecundity

the capacity to reproduce and achieve maximum reproductive output

Hematophagous arthropod

refers to obligate blood-feeding invertebrates

Hemocoel

the body cavity that surrounds all arthropod tissues

Hemocytes

cells that are considered part of the cell-mediated response in arthropods responsible for direct microbe encapsulation, secretion of soluble factors, and transport of essential nutrients to tissues, like macrophages in vertebrate systems

Hemolymph

proteinaceous plasma filling the hemocoel facilitating the transport of hemocytes and secreted factors, similar to that of blood in vertebrate hosts

Hemosomes

iron-rich residual bodies that sequester heme during the digestion of hemoglobin in ticks

Horizontal transmission

refers to the transmission of an organism from one host to a new, susceptible host

Immune deficiency (IMD) pathway

NF-kB-like pathway in arthropods responsible for secreting antimicrobial peptides against Gram-negative organisms

Ixodid tick

refer to hard-bodied ticks

Microbiome

consists of a community of symbiotic microorganisms that play an essential role in the arthropod life cycle

Melanization

a process involving hemocytes where the activation of prophenoloxidase by serine proteases results in the formation of melanin, creating nodules around invading microbes in the hemocoel, ultimately limiting their spread

ML-domain proteins

secreted factors part of an arthropod's immune response that recognize foreign lipids

Nutritional symbiosis

the ability of commensal microbes to provide essential nutrients, such as vitamins or cofactors, to their host

Obligate intracellular bacteria

require host cell processes to reproduce

Parthenogenic

a form of asexual reproduction where embryos can develop in the absence of sperm fertilization

Peritrophic matrix (PM)

amorphous membrane that forms a distinct barrier between the arthropod's blood meal and midgut epithelia. It is comprised mainly of chitin along with a matrix of carbohydrates and proteins. The PM minimizes cellular injury by protecting mucosal surfaces from abrasions or penetration from invading pathogens during blood-feeding

Peptidoglycan recognition proteins (PGRP)

recognize peptidoglycan in the cell wall of bacteria and are a recognition component activating the Toll and IMD pathway in arthropods. PGRP is activated during rickettsial infection in both tick and insect hosts

Reservoir hosts

refers to a host that can harbor a pathogen, serving as a source of infection

Saliva-assisted transmission (SAT)

influence of vector salivary secretions at the arthropod-host interface involved in modulating host homeostasis by regulating innate immune responses, blood coagulation, vasoconstriction, and inflammation

Sialotranscriptome

analyses of an organism's salivary gland transcripts to determine factors associated with infection or its biology

Sympatric

refers to organisms that inhabit the same geographic region

Toll pathway

pathway in arthropods responsible for secreting antimicrobial peptides against Gram-positive organisms and fungi. Although Gram-negative, rickettsiae are known activators of this pathway during arthropod infections

Transstadial transmission

the sequential passage of an organism that is acquired during one life stage and maintained through a molting period and to the next life stage

Transovarial transmission

the passage of an organism from female to offspring via infection of deposited eggs

Vector competence

component of vectorial capacity that depends on vector biology, extrinsic incubation periods, and host immune defenses, ultimately describing the ability of a vector to harbor and transmit a specific organism

Vectorial capacity

refers to a vector's ability to transmit a specific organism at a given time, considering both abiotic and biotic factors

Vertical transmission

the inheritance of an organism throughout a population. It can refer to either transovarial or transstadial transmission

Vitellogenin

molecule synthesized in the fat body, transported through the hemolymph, and engulfed by oocytes of female invertebrates that undergo vitellogenesis. It is responsible for enhancing yolk protein synthesis required for proper embryogenesis by maturing oocytes