https://orcid.org/0000-0003-3361-0780
Abstract
Following several days of blood feeding by larval and nymphal ixodid (hard) ticks, the salivary glands degenerate and are completely replaced in the next life stage. Yet, what happens during the molt of immature argasid (soft) ticks after their rapid and small bloodmeal has remained a mystery. Multiple studies of nymphal Ornithodoros hermsi Wheeler (Acari: Argasidae) ticks infected with the relapsing fever spirochete Borrelia hermsii suggested the salivary glands in these ticks may not disintegrate after feeding. Therefore, cohorts of second-stage O. hermsi nymphs were fed and examined daily after the bloodmeal by fresh dissections and weekly by histological cross-sections of the entire tick. The composition of the salivary glands was typical for argasid ticks in having agranular (Type I) and granular (Type II) acini, the latter being surrounded by a myo-epithelial sheath. In all 197 ticks examined from 1 to 63 days after feeding, morphologically intact salivary glands were present. During apolysis, 5 ticks had extralimital clusters of granular acini adhering to otherwise intact glands. Our observations demonstrate that the salivary glands of nymphal O. hermsi do not disintegrate after feeding and new acini are produced during the molt for incorporation into the existing glands. Cumulatively, these findings suggest a fundamental difference in the transstadial development of argasid and ixodid ticks.
Introduction
Most hematophagous arthropod vectors of pathogens acquire and transmit these agents orally when feeding on vertebrates. For those arthropods with holometabolous development (complete metamorphosis) such as mosquitoes, sandflies, black flies, tsetses, and fleas, only the adults—and for some groups only females—feed on blood and serve as vectors (Smith 1973). In contrast, those species with hemimetabolous development (incomplete metamorphosis), such as reduviid bugs, cimicids, sucking lice and ticks, the immature stages also feed on blood (Balashov 1984) and may acquire, and transmit microorganisms prior to becoming adults. Hard ticks (Ixodidae) generally feed only 3 times, once in each active stage—larva, a single nymph, and adult—and then die, although males in some species may feed multiple times (Sauer et al. 1995). In the absence of transovarial transmission, the larvae and nymphs must acquire a pathogen when feeding for the next stage to be infectious. Whereas soft ticks (Argasidae) also generally feed only once during each immature stage, these ticks develop through multiple nymphal stages, and as adults they may feed and reproduce repeatedly over months to many years (Sonenshine 1991). Thus, soft ticks are long-lived and have many more opportunities to acquire and transmit blood-borne pathogens compared to hard ticks.
Tick salivary glands and the saliva they secrete compose the primary delivery system for pathogens that infect humans and domestic animals when fed upon by these ectoparasites (Binnington and Kemp 1980, Sauer et al. 1995, Šimo et al. 2017). As such, morphological, physiological, and genomic (sialomic) studies of the salivary glands have received much attention to better understand the dynamics of pathogen transmission (Alarcon-Chaidez 2014, Neelakanta and Sultana 2022). When ixodid larvae and nymphs feed, the salivary glands hypertrophy during the several days of attachment and feeding, then disintegrate after engorgement, and are entirely replaced with new glands during the molt to the next life stage (Till 1961, Chinery 1965, Binnington 1978, Kahl et al. 1990). However, the fate of salivary glands in immature argasid ticks after feeding is unknown.
During several studies investigating the relapsing fever spirochete Borrelia hermsii (Davis) (Spirochaetales: Borreliacae) infecting its tick vector, Ornithodoros hermsi Wheeler (Acari: Argasidae), 2 primary observations were made: (i) once colonized, B. hermsii persisted in the ticks’ salivary glands; and (ii) salivary glands were present in all 191 ticks examined 7 to 448 days after nymphal feeding (Table 1) (Schwan and Hinnebusch 1998, Raffel et al. 2014, Schwan et al. 2020, Schwan 2021). These observations suggested a possible fundamental difference in the persistence of the salivary glands in nymphal soft ticks after feeding and during their molt, in contrast to the degeneration of these glands in hard ticks.
Presence of intact salivary glands in nymphal Ornithodoros hermsi infected with Borrelia hermsii examined intermittently 7–448 days after an infectious bloodmeal
| Study | Days examineda | Intact salivary glands | SG +/ SG | % SG infectedb | Reference |
|---|---|---|---|---|---|
| 1 | 33–144 | 41/41 | 41/41 | 100% | Schwan and Hinnebusch 1998 |
| 2 | 7–448 | 103/103 | 81/103 | 78.6% | Raffel et al. 2014 |
| 3 | 14–203 | 16/16 | 9/16 | 56.3% | Schwan et al. 2020 |
| 4 | 7–260 | 31/31 | 29/31 | 93.5% | Schwan 2021 |
| Totals | 191/191 | 160/191 | 83.3% |
| Study | Days examineda | Intact salivary glands | SG +/ SG | % SG infectedb | Reference |
|---|---|---|---|---|---|
| 1 | 33–144 | 41/41 | 41/41 | 100% | Schwan and Hinnebusch 1998 |
| 2 | 7–448 | 103/103 | 81/103 | 78.6% | Raffel et al. 2014 |
| 3 | 14–203 | 16/16 | 9/16 | 56.3% | Schwan et al. 2020 |
| 4 | 7–260 | 31/31 | 29/31 | 93.5% | Schwan 2021 |
| Totals | 191/191 | 160/191 | 83.3% |
aDays after tick feeding.
bMajority of those salivary glands not infected were examined during the first 2 wk after feeding.
Presence of intact salivary glands in nymphal Ornithodoros hermsi infected with Borrelia hermsii examined intermittently 7–448 days after an infectious bloodmeal
| Study | Days examineda | Intact salivary glands | SG +/ SG | % SG infectedb | Reference |
|---|---|---|---|---|---|
| 1 | 33–144 | 41/41 | 41/41 | 100% | Schwan and Hinnebusch 1998 |
| 2 | 7–448 | 103/103 | 81/103 | 78.6% | Raffel et al. 2014 |
| 3 | 14–203 | 16/16 | 9/16 | 56.3% | Schwan et al. 2020 |
| 4 | 7–260 | 31/31 | 29/31 | 93.5% | Schwan 2021 |
| Totals | 191/191 | 160/191 | 83.3% |
| Study | Days examineda | Intact salivary glands | SG +/ SG | % SG infectedb | Reference |
|---|---|---|---|---|---|
| 1 | 33–144 | 41/41 | 41/41 | 100% | Schwan and Hinnebusch 1998 |
| 2 | 7–448 | 103/103 | 81/103 | 78.6% | Raffel et al. 2014 |
| 3 | 14–203 | 16/16 | 9/16 | 56.3% | Schwan et al. 2020 |
| 4 | 7–260 | 31/31 | 29/31 | 93.5% | Schwan 2021 |
| Totals | 191/191 | 160/191 | 83.3% |
aDays after tick feeding.
bMajority of those salivary glands not infected were examined during the first 2 wk after feeding.
Robinson and Davidson’s (1913) description of the salivary glands in adults of the soft tick Argas persicus (Oken) (Acari: Argasidae) included the following: “In the resting phase after engorgement, they [the salivary glands] may assume quite insignificant proportions, though they rarely show the extreme degree of degeneration which is exhibited by the Ixodidae.” Guirgis’s (1971) extensive histological investigation of the closely related soft tick Argas arboreus Kaiser, Hoogstraal & Kohls (Acari: Argasidae) included the salivary glands of nymphal ticks and found there was some shrinkage of the salivary gland cells by the end of engorgement but they “regain their normal appearance in the first few days of the postfeeding period.” In Till’s (1961) introduction to her work on the salivary glands of the hard tick Rhipicephalus appendiculatus Neumann (Acari: Ixodidae), she stated that following engorgement by hard ticks the salivary glands degenerate, while in argasids these glands go through a “reduction,” but no other information was given. True (1932) examined salivary glands of Ornithodoros coriaceus Koch (Acari: Argasidae) and noted “In specimens which had fed recently, the salivary glands were uniformly larger than in those which had not fed for some time,” but no ticks were followed after feeding or during the molt. Therefore, to address the question of what happens to the salivary glands of immature O. hermsi following a bloodmeal, cohorts of nymphal ticks were fed and examined sequentially to determine if their salivary glands disintegrated or were retained during the molt, the latter being our hypothesis based on previous observations (Schwan and Hinnebusch 1998, Raffel et al. 2014, Schwan et al. 2020, Schwan 2021), and what we now show herein.
Methods and Materials
Tick Specimens
Ornithodoros hermsi SIS ticks were from a colony at the Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana, established with a single, engorged, uninfected female tick collected from Siskyou County, California. The colony was maintained by feeding ticks on 4- to 7-day-old laboratory mice (Mus musculus) and kept between feedings at room temperature (~21 °C), ambient light and 85% relative humidity as described (Schwan 2021).
Gross Examination of Tick Salivary Glands
A cohort of second-stage O. hermsi nymphs was fed on mice and individual ticks were examined for most of 48 days after engorgement; no ticks were sampled on day 28 and days 33–38. A total of 2–7 ticks were selected randomly each day and examined with a Zeiss Stemi SV11 stereoscopic binocular microscope (Carl Zeiss Microscopy, LLC, White Plains, NY) to check for a developing opacity of the integument that indicated the molting process had begun. Ticks were then placed in a drop of phosphate-buffered saline (PBS) on a glass microscope slide and while viewed with the microscope, the dorsal integument was removed with a pair of fine-tipped forceps. If the tick had begun apolysis—the separation of the old exoskeleton from the underlying epidermis (Jenkin and Hinton 1966)—this was noted and the old outer integument was removed first before entering the body cavity through the newly forming cuticle. The 2 salivary glands were identified in situ, removed and placed in fresh PBS on another microscope slide for examination at higher magnification with a Nikon Eclipse E800 microscope and differential interference contrast (DIC) illumination (Nikon Instruments, Inc., Melville, NY). Some specimens were also viewed with the same microscope using UV illumination and a DAPI filter cube (UV-2E/C; exciter wavelength 330–380, barrier wavelength 420). Images were acquired with a DXM1200c digital camera and ACT-1C software, version 1.0.1.5 (Nikon Instruments).
Histological Examination of Ticks
Another cohort of second-stage nymphs was fed on mice and 1, 2, 3, 5, and 9 wk (7–63 days) after engorgement, 2 or 3 ticks were fixed in Bouin’s solution (Sigma HT, 10132-1L; Sigma-Aldrich, St. Louis, MO). The specimens were embedded in paraffin and 5 µm serial cross-sections of the entire tick from rostral to caudal ends were prepared and placed in series on glass microscope slides, stained with hematoxylin and eosin, and mounted with Cytoseal-XYL 83124 (Epredia, Kalamazoo, MI) and 24 × 50 mm glass coverslips. One unfed second-stage nymph was also included for histological analysis. All cross-sections (N = 730) were examined with the Nikon Eclipse E800 microscope and again with a Leitz Ortholux microscope. Images were acquired with the Leitz microscope, OMAX A35180U3 digital camera, ToupView, version macOS 1.0.9229 (Top View, Irvine, CA) and Adobe Photoshop Elements 2018 software packages (Adobe Inc., San Jose, CA).
Scanning Electron Microscopy
Dissected salivary glands from adults of 4 species of Ornithodoros ticks were fixed overnight at 4 °C with 2% paraformaldehyde and 2.5% glutaraldehyde buffered with 100 mmol/L sodium cacodylate. Samples were washed with 100 mmol/L sodium cacodylate, postfixed with 1% OsO4 for 1 h, and dehydrated in a graded ethanol series. Specimens were critical point-dried under CO2 with a Bal-Tec cpd 030 drier (Balzers), mounted on aluminum studs, and coated with 125 Å of iridium in an IBS/TM200S ion beam sputterer (South Bay Technologies, currently under Ted Pella, Redding, CA) before viewing at 5 kV on a S-4500 field emission scanning electron microscope (Hitachi High Technologies, Inc., Tokyo Japan).
Sections for Light Microscopy
Additional dissected salivary glands from O. hermsi were fixed and dehydrated as described above and embedded in Spurr’s resin. Semi-thin 0.25 mm sections were cut and stained with Toluidine Blue and Basic Fuchsin (Electron Microscopy Sciences, Hatfield, PA) and examined by light microscopy.
Ethics Statement
The RML, NIAID, NIH, Animal Care and Use Committee approved study protocols for feeding ticks and maintaining the colony on mice (2009-87, 2012-70, 2015-82, 2018-062). All work was done in adherence to the institution’s guidelines for animal husbandry and followed the guidelines and basic principals in the Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals, United States Institute of Laboratory Animal Resources, National Research Council.
Results
Composition of the Salivary Glands
The salivary glands of O. hermsi were typical for argasid ticks in having agranular (Type I) and granular (Type II) acini (=alveoli). The less abundant agranular acini were restricted to the medial-anterior portion of the glands (Fig. 1A) and auto-fluoresced more brightly than did the granular acini when fresh, unfixed glands were illuminated with UV light and viewed with the DAPI filter (Fig. 1B). The agranular acini were closely aligned to the main salivary duct to which each acinus was directly connected via a short efferent duct (Fig. 1C), as in other argasid ticks (Sonenshine and Gregson 1970, Roshdy 1972, Coons and Alberti 1999). Spiral thickenings in the cuticular lining of the main and efferent ducts were also evident (Fig. 1C). The granular acini were primarily connected by multiply branched ducts before joining the main salivary duct. These acini contained secretory granules of different size and density (Fig. 1D) but were not identified with specific stains or immunological reagents.
Ornithodoros hermsi salivary glands are comprised of agranular (Type I*) and granular (Type II**) acini. Gland visualized with DIC illumination (A), UV illumination and DAPI filter (B), agranular acini with efferent duct joining main salivary duct (C), and stained section showing granular (lower left) and agranular acini (upper right) (D). Panels A and B, 640 µm wide; panels C and D, 170 µm wide.
The granular but not agranular acini were surrounded by a myo-epithelial sheath, which was translucent and not at first obvious when viewed by bright-field microscopy. However, the sheath was visible when viewing the perimeter of freshly dissected glands with DIC illumination at 100× magnification (Fig. 1A, above**). Therefore, we applied SEM to view dissected salivary glands of O. hermsi and 3 additional species of Ornithodoros from remnant colonies at RML. Besides O. hermsi, only the granular acini were surrounded by a sheath in the salivary glands of Ornithodoros turicata (Dugès) (Acari: Argasidae), Ornithodoros tholozani (Laboulène & Mégnin) (Acari: Argasidae) (Fig. 2), and Ornithodoros zumpti Heisch & Guggisberg (Acari: Argasidae) (not shown). For intact specimens, only the agranular acini were visible as the sheath presented an electron-reflective barrier concealing the granular acini within (Fig. 2C and D), which were visible when the myo-epithelial sheath was broken (Fig. 2A and B).
SEM salivary gland images of adult O. hermsi (A), O. turicata (B, C), and O. tholozani (D) showing the myo-epithelial sheath (*) surrounding the granular acini. Fractured glands with a torn sheath uncover the granular acini (A, B), while only the band of agranular acini (**) are visible with intact glands (C, D).
Gross Structure of the Salivary Glands
The salivary glands from 184 nymphal O. hermsi ticks were examined on 41 of 48 days after engorgement (Table 2). In every tick, 2 intact salivary glands were present and removed with fine-tipped forceps for microscopic examination. During the first 22 days, all salivary glands appeared the same with the sheath-bound compartmentalization of the granular acini separated from the fewer agranular acini (Fig. 1A). However, once the molting process of this cohort began on day 23 after feeding, 5 of 18 ticks examined during apolysis and prior to the completion of the molt had clusters of granular acini immediately adjacent to the intact but somewhat irregular shaped salivary glands that still contained both agranular and granular acini (Fig. 3). No agranular acini were observed in these extralimital clusters. Additionally, in 11 ticks that had recently molted to the third nymphal stage (Table 2), only normal, intact salivary glands were seen. These observations demonstrated that the salivary glands did not degenerate during the molt, but rather new acini were produced for incorporation into the existing glands.
Examination of salivary glands in Ornithodoros hermsi second-stage nymphs on days 1–48 after feeding
| Days after feeding | No. ticks examined | No. in apolysis | No. molted |
|---|---|---|---|
| 1–22 | 82 | 0 | 0 |
| 23–32a | 46 | 5 | 2 |
| 39–48 | 56 | 13 | 9 |
| Totals | 184 | 18 | 11b |
| Days after feeding | No. ticks examined | No. in apolysis | No. molted |
|---|---|---|---|
| 1–22 | 82 | 0 | 0 |
| 23–32a | 46 | 5 | 2 |
| 39–48 | 56 | 13 | 9 |
| Totals | 184 | 18 | 11b |
aNo ticks examined day 28.
bAll third-stage nymphs.
Examination of salivary glands in Ornithodoros hermsi second-stage nymphs on days 1–48 after feeding
| Days after feeding | No. ticks examined | No. in apolysis | No. molted |
|---|---|---|---|
| 1–22 | 82 | 0 | 0 |
| 23–32a | 46 | 5 | 2 |
| 39–48 | 56 | 13 | 9 |
| Totals | 184 | 18 | 11b |
| Days after feeding | No. ticks examined | No. in apolysis | No. molted |
|---|---|---|---|
| 1–22 | 82 | 0 | 0 |
| 23–32a | 46 | 5 | 2 |
| 39–48 | 56 | 13 | 9 |
| Totals | 184 | 18 | 11b |
aNo ticks examined day 28.
bAll third-stage nymphs.
Paired salivary glands from an apolytic O. hermsi nymph 29 days after feeding. Anterior end of each gland to left. Note the extralimital clusters of granular acini (*) (A, B) and the row of agranular acini incorporated with the intact gland (B, under black bar). Panels 640 µm wide.
Histological Examinations of the Salivary Glands
Serial cross-sections of 1 unfed and 13 fed nymphal O. hermsi ticks examined 1–9 weeks after engorgement all showed morphologically intact salivary glands (Fig. 4). For consistency of position, images of the salivary glands shown were taken from the cross-section of each tick having the esophagus in the vertical center of the synganglion. The 2 specimens fixed at 5 wk after feeding were in the final stages of apolysis with the old integument widely separated from the underlying, newly formed cuticle (Fig. 5A), yet the salivary glands were present and structurally intact (Fig. 5B). In these ticks, there were granular acini that appeared as lumps bulging from the usual elliptical shape of the glands seen in cross-sections of the other specimens (Fig. 5B). Whether these acini represented the extralimital clusters seen in the fresh apolytic nymphs is not known. However, the histological examinations supported the gross observations that the salivary glands of nymphal O. hermsi were retained after feeding.
Histological sections of salivary glands in O. hermsi nymphs before feeding (A) and 1 wk (B), 2 wk (C), 3 wk (D), 5 wk (E), and 9 wk (F) after feeding. All images 220 µm wide with dorsal aspect above and medial aspect to right. Specimen examined at 9 wk (F) a recently molted female.
Histological sections of a molting O. hermsi nymph 5 wk after feeding showing the wide separation of the old outer (*) and new inner (**) cuticle (A) and the morphologically intact salivary gland (B). Note the bulbar extension of granular acini in the lower left (*). Panel A, 430 µm wide; Panel B, 220 µm wide. Panel B same as Fig. 3E.
Discussion
The daily dissections and weekly serial sections of nymphal O. hermsi after engorgement demonstrated the salivary glands did not disintegrate during the ticks’ transstadial development to the next nymphal stage. Although the number of ticks examined during apolysis was small (18/184; Table 2), 5 of these ticks had clusters of granular acini adhering externally to intact salivary glands, which were never observed with glands removed from ticks prior to or after their molt. Therefore, we conclude that during the molt, new acini were produced for incorporation into the existing salivary glands. Cumulatively, our finding of intact salivary glands in all nymphal O. hermsi examined after feeding (Tables 1 and 2), suggests a fundamental difference in the transstadial development of argasid and ixodid ticks.
Our examination of freshly dissected O. hermsi salivary glands demonstrated that the granular but not agranular acini were surrounded by a sheath or myo-epithelial membrane. This tissue has received little attention and was not mentioned with descriptions of the salivary glands in O. coriaceus (True 1932) and Ornithodoros kelleyi Cooley & Kohls (Acari: Argasidae) (Sonenshine and Gregson 1970). Coons and Alberti (1999) were first to comment on this structure: “A sheath composed of flattened epithelial cells surrounds the salivary glands of Argasidae (Coons, personal observations). It is absent in the Ixodidae.” Bowman and Sauer (2004) also stated “The salivary glands of argasid ticks, in contrast to ixodid ticks, are surrounded by a myo-epithelial sheath.” Mans (2002) and Mans and Neitz (2004) first showed this myo-epithelial sheath with images taken by low magnification scanning electron microscopy (SEM); the entire salivary glands of Ornithodoros savignyi (Audouin) (Acari: Argasidae) are each surrounded by this sheath. Mastropaolo et al. (2011) also applied low magnification SEM and found that only the granular acini in the salivary glands of nymphal Otobius megnini (Dugès) (Acari: Argasidae) are surrounded by the sheath. Because this sheath is very thin and translucent, it was difficult to see when examining salivary glands with a standard, bright-field microscope. However, by SEM the myo-epithelial sheath was clearly visible and showed that besides O. hermsi, only the granular acini were surrounded by the sheath in the salivary glands of O. turicata, O. zumpti, and O. tholozani (Fig. 2), all members of the subgenus Pavlovskyella. This contrasts with salivary glands of O. savignyi in the subgenus Ornithodoros that are surrounded entirely by this sheath (Mans 2002, Mans and Neitz 2004). These differences suggest that the myo-epithelial sheath may be an additional morphological character to examine among a greater diversity of soft ticks to further our understanding of the evolution of the Argasidae (Mans 2023).
With the production of new acini during the nymphal molt, the question arises as to how these structures are produced and then incorporated into the existing salivary glands? While we only observed granular acini in the extralimital clusters during apolysis, we assume that some agranular acini were also produced but possibly in fewer numbers and not visible in the sample preparations we examined. In molting larvae and nymphs of the ixodid ticks R. sanguineus, Haemaphysalis spinigera Neumann (Acari: Ixodidae), and Ixodes ricinus (Linnaeus) (Acari: Ixodidae), Till (1961), Chinery (1965), and Kahl et al. (1990), respectively, each described the degeneration of the salivary glands following engorgement and their replacement with new acini and glands arising from epithelial cells associated with the cuticle at the ends of remaining salivary ducts. If we are correct about the synthesis of new acini during the molt of O. hermsi, then such progenitor cells exist and await description. Additionally, the new granular acini must be integrated into the existing salivary glands and surrounded by the myo-epithelial sheath, which therefore would also have to grow during the molt. Such reorganization may have been in progress with salivary glands from the apolytic nymph (Fig. 3), as some extensions of the myo-epithelial sheath were visible below the anterior ends of each gland. Additionally, while not measured in the present study, clearly the salivary glands in O. hermsi increase in size as these ticks grow larger in their life cycle from tiny 0.6- to 0.7-mm-long larvae to 3- to 5-mm-long adults (Wheeler 1935, Kohls et al. 1965, Schwan, personal observation). The salivary glands in adult Argas hermanni Audouin (Acari: Argasidae) are larger than in nymphal ticks, and both Type I and Type II acini are larger as well (Khalil et al. 1993). As the salivary glands are replaced during the development of R. sanguineus and I. ricinus from larvae to nymphs and nymphs to adults, the number of acini increase (Till 1961, Kahl et al. 1990). Further studies await to determine changes in size, number, and acinar composition of the salivary glands during the transstadial development of O. hermsi and other argasid species.
Several studies examined salivary glands and specific acini of argasid ticks before and shortly after feeding but observations extended at most only 8–10 days after the ticks fed (Guirgis 1971, Chinery 1974, El Shoura 1985, 1987, Mans 2002). These investigations showed some enlargement of the salivary glands immediately after engorgement with various degrees of degranulation of Type II granular acini, but after several days the gross structure of the glands returned to their normal appearance as they were before feeding and were not examined later. However, it is unclear if nymphs or adults were examined in some of these studies. We found only one investigation that addressed possible disintegration of salivary glands in nymphal soft ticks after feeding. Stricker (1993) examined the one-host, spinose ear tick O. megnini at 2, 5, and 8 days after engorged nymphs were removed from experimentally infested sheep but ticks were not sampled later or during the molt. During the 8 days after detachment, the granular acini progressively disintegrated while the agranular acini did not. After the molt, agranular acini were present in adults but only remnants of the granular acini remained and were small and wrinkled compared to the nymphal glands (Stricker 1993, Mastropaolo et al. 2011). These investigators concluded that the granular acini were not needed by the adult O. megnini as this stage of these ticks live off-host and do not feed.
The cohort of nymphal O. hermsi examined daily did not begin apolysis until 23 days after feeding. In our previous studies, B. hermsii began colonizing salivary glands 7 days (the earliest time examined) after nymphal O. hermsi fed on spirochetemic mice (Schwan 2021). By 3 wk after feeding, the majority of ticks had infected salivary glands (Raffel et al. 2014, Schwan 2021). Therefore, B. hermsii may penetrate intact salivary glands prior to the growth of new acini before the molt. The distance for dissemination of spirochetes from the midgut to salivary glands during this time may be quite short, possibly less than 2 µm, and shorter than the length of a single spirochete (Barbour and Schwan 2018), as the midgut wall lies immediately adjacent to and in contact with the salivary glands (Fig. 4B-D). Borrelia hermsii infects only the granular acini (Fischer and Schwan 1999, Policastro et al. 2013), like African swine fever virus in Ornithodoros porcinus porcinus Walton (Acari: Argasidae) (Kleiboeker et al. 1998) and the protozoan parasite Theileria parva (Theiler) (Piroplasmida: Theileriidae) that infects only the e cells of Type III granular acini in R. appendiculatus (Fawcett et al. 1981, 1982). The specificity of B. hermsii for granular acini may be the essential pathway for transmission of spirochetes by these fast-feeding ticks, given the role the secretions of these acini play for feeding (Mans et al. 2004). In contrast, the agranular acini are primarily involved in secreting salts onto the hypostome for hygroscopic absorption of water from unsaturated water vapor, which is then swallowed and essential for survival of ticks during long periods of fasting while off the host (Balashov 1972, Needham and Teel 1986, 1991, Kahl et al. 1990). Infecting agranular acini of O. hermsi would likely be a dead end for B. hermsii.
Lastly, the question arises as to why immature hard ticks have evolved with the complete destruction and replacement of the salivary glands in the next life stage while the argasid ticks have not. One possible explanation may lie in the energy cost in molting in relation to the size of the bloodmeal imbibed during feeding. Most nutrients in the bloodmeal, primarily hemoglobin, ingested by immature ticks are utilized in the production of new cuticle for the next larger instar (Sonenshine 1991). But clearly some energy must be directed to the synthesis of entirely new salivary glands (the ixodids) or the production of new acini to be incorporated into the existing glands (the argasids). Determining the total amount of blood ingested by ixodid ticks that feed for many days is problematic, given the salivary secretion of salts and water back to the host and the excretion of feces while attached (Kaufman 1989, Sonenshine 1991). Thus, the increase in body weight of an ixodid tick before and immediately after detachment certainly underrepresents the total amount of blood ingested (Koch and Sauer 1984). In contrast, an accurate measurement of the total weight gained with a bloodmeal consumed by fast-feeding argasids that do not excrete coxal fluid while on the host is easy to measure (Balashov 1972, Sonenshine 1991). Such was the case for O. hermsi when second-stage nymphs with an average unfed weight of 0.498 mg ingested an average of 2.827 µl of blood (McCoy et al. 2010). For nymphal Ixodes persulcatus Schulze (Acari: Ixodidae) with an average weight of 0.21 mg before feeding, a bloodmeal size based on the ticks’ weights after feeding and the amount of hemoglobin in the ticks and excreted feces yielded an estimated average volume of 15.861 µl per tick (Balashov 1972), which is nearly 6 times the amount of blood consumed by O. hermsi. Thus, we speculate that one of the consequences of a much smaller bloodmeal consumed by immature argasid ticks is the impact of reduced nutrients available for growth of new tissues. The retention rather than destruction of the existing salivary glands during the molt of argasid nymphs may have evolved as an adaptation to rapid feeding and smaller bloodmeals.
In conclusion, the retention and growth of the salivary glands of nymphal O. hermsi through their transstadial development to the next active stage in the life cycle contrasts with the paradigm of complete degeneration and replacement of these glands established in ixodid ticks. Yet, we caution that O. hermsi is only one of approximately 200 species of argasid ticks (Guglielmone et al. 2010, Dantas-Torres 2018, Mans et al. 2019) with no other species having been examined as we have done herein. Therefore, we hope our findings will prompt other investigators to pursue further studies concerning the development of salivary glands in other species of molting argasid ticks. A better understanding of the growth and morphogenesis of these glands should also further our knowledge of spirochete-tick specificity (Schwan 1921) that results, in part, from the persistent infection of the salivary glands during the transstadial development of immature argasid ticks.
Acknowledgments
We thank members of the Animal Care and Use Committee for oversight and approval to feed ticks on mice, and staff of the Rocky Mountain Veterinary Branch for providing mice for the maintenance of our tick colony.
Funding
This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Author contributions
Tom Schwan (Conceptualization [lead], Formal analysis [lead], Funding acquisition [equal], Investigation [lead], Methodology [equal], Project administration [lead], Resources [equal], Supervision [lead], Validation [equal], Visualization [equal], Writing—original draft [lead], Writing—review & editing [equal]), Elizabeth Fischer (Conceptualization [supporting], Data curation [supporting], Formal analysis [supporting], Funding acquisition [equal], Investigation [supporting], Methodology [equal], Project administration [supporting], Resources [equal], Validation [equal], Visualization [equal], Writing—review & editing [equal]), and Daniel Long (Conceptualization [supporting], Data curation [supporting], Formal analysis [supporting], Funding acquisition [equal], Investigation [supporting], Methodology [equal], Project administration [supporting], Resources [equal], Validation [equal], Visualization [equal], Writing—review & editing [equal])
