Ticks and Tick-borne Diseases 5 (2014) 569–574
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Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Salivary gland degeneration and ovarian development in the Rocky Mountain wood tick, Dermacentor andersoni Stiles (Acari: Ixodidae). I. Post-engorgement events Shahid A.K.M. Ullah 1 , W. Reuben Kaufman ∗ Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6E 2E9
a r t i c l e
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Article history: Received 5 December 2013 Received in revised form 22 February 2014 Accepted 25 March 2014 Available online 24 June 2014 Keywords: Ixodid ticks Dermacentor andersoni Amblyomma hebraeum Salivary gland degeneration Ovary development Ovary vitellin content
a b s t r a c t It is well known that female ixodid ticks undergo salivary gland degeneration and the greater part of ovarian development following engorgement. The process has been particularly well studied in Amblyomma hebraeum, including the hormonal control of these processes. The purpose of this study, and the second one in this series, is to compare the processes in Dermacentor andersoni with those of A. hebraeum. A major difference between the two species is that virgin female D. andersoni feed to a much higher fed-tounfed weight ratio than do virgin female A. hebraeum, the former achieving an average of 67× the unfed weight in this study, the latter normally achieving an average of only about 10–15× the unfed weight. We show here that although engorged virgins (defined here as those that exceed a fed-unfed weight ratio of 50, even though they do not detach from the host spontaneously) degenerate their salivary glands and develop their ovaries, these processes proceed more slowly in virgins that they do in mated females, as is the case for A. hebraeum. © 2014 Elsevier GmbH. All rights reserved.
Introduction Female ixodid ticks, depending on species and stage of life cycle, require 5–10 days or so to engorge fully. During this period, the salivary glands (SGs) undergo an enormous degree of growth and development. For example, the maximum fluid secretory rate of isolated SGs from partially fed D. andersoni peaks at about 54× the rate observed for unfed ticks (Kaufman, 1976), a process that is controlled in A. hebraeum by a haemolymph-borne factor (Coons and Kaufman, 1988). However, within only about 4 days following engorgement, the SGs of A. hebraeum undergo autolysis; ovarian development, the early stages of which have begun during feeding, is completed within about 10–12 days post engorgement, when oviposition begins (Lunke and Kaufman, 1993; Friesen and Kaufman, 2002). The phenomenon of SG degeneration, based on histological data, has been noted since at least the 1940s (Vitzthum, 1943, quoted by Till, 1961), and subsequently well established by the 1960s (Till, 1961). The hormonal control of SG degeneration was
first demonstrated by Harris and Kaufman (1981) in A. hebraeum; they named the hormone ‘tick salivary gland degeneration factor’. A few years later, tick salivary gland degeneration factor was shown to be an ecdysteroid hormone (Harris and Kaufman, 1985). Like many ixodid ticks, A. hebraeum must copulate before they can engorge. The approximate maximum fed-to-unfed weight ratio achieved by most virgin A. hebraeum is only about 10, a ratio that is too low to trigger SG degeneration or ovarian development (Kaufman, 2007). But virgin D. andersoni normally feed to a much higher weight ratio than do A. hebraeum (Kaufman, 2007). Even though such large virgins do not usually detach spontaneously from the host, they are at a size sufficient to trigger SG degeneration and ovarian development. The objective of this study was to determine the dynamics of SG degeneration and ovarian development in normally engorged mated D. andersoni and in nominally “engorged” virgins (those that achieve >50× the unfed weight, even though they do not detach spontaneously), and to compare these dynamics with those previously established for A. hebraeum. Materials and methods
∗ Corresponding author at: Cormorant Crescent, Salt Spring Island, BC, Canada V8K 1G8. Tel.: +1 250 931 0033. E-mail address: [email protected] (W.R. Kaufman). 1 Present address: Unit 101, 10710, 85 Avenue NW, Edmonton, Alberta, Canada T6E 2K8. http://dx.doi.org/10.1016/j.ttbdis.2014.03.012 1877-959X/© 2014 Elsevier GmbH. All rights reserved.
Ticks The adult D. andersoni ticks used in this study were provided by Dr. Tym Lysyk (Agriculture and Agri-Food Canada, Lethbridge Research Centre, Canada), mean female unfed weight of
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6.47 ± 0.13 mg (n = 260), and by Dr. Glen Scoles, (Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington and ARS, USDA), mean female unfed weight of 4.67 ± 0.06 mg (n = 328). As we frequently compare the results of this study with those done previously using A. hebraeum, the mean unfed weight for a large sample of the A. hebraeum females from our recent colony was 30.0 ± 0.6 mg (n = 192). After arriving at our laboratory, ticks were maintained in darkness, at 7–9 ◦ C, and held over saturated KNO3 (RH ∼95%). The day before putting them on a New Zealand white rabbit (SPF; Charles River, Senneville, Quebec, Canada), the ticks were transferred to a humid, 27 ◦ C incubator. A cloth-covered foam ‘backpack’ (∼12 cm × 8 cm × 2.5 cm) was glued to the shaven back of the rabbit with a latex adhesive (Roberts 8502 Latex, Bramalea, Ontario, Canada). In order to determine the ultimate fed-to-unfed weight ratio of each experimental tick, the ticks were coded prior to feeding by glueing a coloured silk thread to a leg, and then weighed. Equal numbers of males and females were introduced together to the backpack for experiments requiring mated females; only females were added to the backpack for experiments requiring virgin females. Mated females normally engorged and detached spontaneously after 4–6 days of feeding. Virgin females that were allowed to feed on the host for 21 days (the maximum allowed by our animal use protocol) and which exceeded about 50× the unfed weight, were defined as “fully engorged”, even though they did not detach from the host spontaneously. After collecting the ticks from the host, they were rinsed with water, dried with tissue paper, weighed, and stored (27 ◦ C) individually in cloth-covered plastic vials until dissection. Our animal-use protocol was approved by the Biosciences Animal Policy and Welfare Committee of the University of Alberta. This committee functions according to the current guidelines established by the Canadian Council on Animal Care. Assay for salivary fluid secretory competence The technique used was based on that described by Harris and Kaufman (1984). Briefly, ticks were glued ventral surface down to a disposable Petri dish and cooled in a refrigerator for about 30 min. They were then bathed in modified Hank’s saline (per litre: NaCl 11.5 g, d-glucose 1.6 g, KCl 0.4 g, phenol red 0.01 g; the pH was adjusted to 7.2) and the SGs and ovary dissected out. The tissues were transferred to TC medium 199 made with Hank’s salts (Sigma Chemical Company, St. Louise, MO, USA). Before dissolving a package of the powdered medium in 1 L milliQ (Millipore) water, we added 2.09 g of the buffer, morpholinopropanesulphonic acid (MOPS), plus 2.1 g NaCl; the pH was adjusted to 7.2. The SGs and ovary were dissected free of extraneous tissues. The main salivary duct was ligated with a fine strand peeled from silk thread. Each gland was transferred to a hydrophobic surface and extraglandular fluid was removed by gentle application of a piece of filter paper. The gland was then weighed to the nearest 10 g on a 5-place electronic balance and immediately transferred to a bathing medium of TC medium 199 containing freshly prepared dopamine (10 M; Sigma Chemical Co.). The bathing medium was slowly agitated with a magnetic stir-bar during the 15-min incubation period. The increase in wet weight per gland between 0 and 15 min was used as an index of salivary fluid secretory competence (Harris and Kaufman, 1984). Dopamine is probably the natural neurotransmitter for salivary fluid secretion, and 10 M is a supra-maximal concentration (Kaufman, 1976). Ovary maturation assays The ovary was dissected out and submerged in TC medium 199, and the lengths of 10 apparently largest oocytes were measured
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salivary fluid transport (mg/gland/15 min)
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days post engorgement Fig. 1. Rate of salivary fluid uptake in engorged (mated and virgin) D. andersoni females as a function of days post engorgement. Mean, SEM, and N are indicated for each bar. Among the mated ticks, a significant loss of fluid secretory competence occurred throughout 4 days post engorgement (in all cases p = 0.000 in comparison to day 0). Among the virgin ticks, a significant loss of fluid secretory competence began only on day 4 post engorgement (p = 0.000 in comparison to day 0).
using a dissecting microscope fitted with an ocular micrometre. The ovary was gently blotted with filter paper and weighed to the nearest 10 g. The ovary was then homogenized in 1 ml milliQ water, frozen quickly on dry ice, and stored at −20 ◦ C in small polyethylene microfuge tubes. For the spectrophotometric analysis of vitellin content, ovary homogenates were thawed and diluted with milliQ water to a final volume of 2 ml. After gentle vortexing, the homogenates were centrifuged at 12,000 rpm for 10 min. The supernatant was collected and stored at −20 ◦ C. For the final analysis, samples and blank were thawed, and the absorbance at 400 nm (specific for the hem moiety of vitellin) and 500 nm (non-specific for hem) were measured on a spectrophotometer (Ultrospec 3300 pro, Biochrom Ltd, Cambridge, England). The difference between the 2 absorbance readings was accepted as a measure of vitellin content of the sample and was normalized to ovary weight (expressed as 400 nm 500 nm per gram ovary; Kaufman et al., 1986; Seixas et al., 2008). Transmission electron microscopy (TEM) of salivary glands The technique was based on the optimized protocol used in the Microscopy Unit, Department of Biological Sciences. Briefly, SGs were dissected out under Hank’s saline. The large tracheae were dissected away, and the glands were fixed at 4 ◦ C for at least 2 h in a solution containing 2.5% glutaraldehyde, 2% paraformaldehyde, 0.1 M phosphate buffer (pH 7.3). After fixation, the tissue was washed 4 times with 0.1 M phosphate buffer (2 quick changes followed by 2 changes 10 min apart). The glands were post-fixed with 1% OsO4 in 0.12 M cacodylate buffer, pH 7.2. The glands were washed again with 3 changes of 0.1 M phosphate buffer over 30–60 min. The glands were then dehydrated through a graded ethanol series (50%, 70%, 90%, 15 min each change), followed by 3 changes of absolute ethanol. The glands were incubated overnight in 1:1 ethanol:low viscosity spur resin (Electron Microscopy Sciences, Hatfield, PA, USA). This was followed by 2 changes of pure spur resin, a few hours apart. The glands in resin were then transferred to moulds and held at 70–80 ◦ C for 16–18 h. Tissue blocks were sectioned (70–90 nm thick) using an Ultracut E Reichert-Jung Ultramicrotome. Sections were transferred to copper grids (200 mesh). The sections were stained with 4% uranyl acetate for 25 min and counterstained in lead citrate (per 50 ml solution: 1.33 g lead
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Fig. 3. Fluid secretory type III acinus of (A) a 2-day PE-M (post engorgementmated) D. andersoni female; 96× the unfed weight and (B) a 2-day PE-V (post engorgement-virgin) female; 102× the unfed weight. In both mated and virgin female SGs, autophagic vacuoles are apparent, although they are still more prominent in the mated female SG. AV, autophagic vacuoles; M, mitochondria; L, salivary fluid secretory labyrinth. Fig. 2. Fluid secretory type III acinus of (A) a 0-day PE-M (post engorgementmated) D. andersoni female; 93× the unfed weight and (B) a 0-day PE-V (post engorgement-virgin) female, 81× the unfed weight. In both mated and virgin female SGs, autophagic vacuoles are apparent, although they appear to be more prominent in the mated female SG. AV, autophagic vacuoles; M, mitochondria; L, salivary fluid secretory labyrinth; H, haemolymph.
citrate, 1.76 g sodium citrate in double distilled, vacuum-filtered H2 O, pH-adjusted to 12.0 by 0.01 N NaOH) for 2 min. Grids were examined by TEM (Philips Morgagni 268 and Philips 410, operating at 80 kV), and photomicrographs were taken with a Gatan Orius CCD camera. Statistics Unless otherwise stated, all data are reported as mean ± SEM (N), using Microsoft Excel software (Microsoft Office 2007). Statistical significance was determined by student’s t-test using STATA 10.0 (StataCorp, TX, USA) on a Macintosh Computer. When analyzing the effect of time post engorgement, the comparator group was always day 0. Other comparisons will be specified within figure legends. Results The fed/unfed weight ratio for engorged D. andersoni females used in this experiment was in the range of 50–100 [average for mated: 87 ± 7 (n = 62); average for virgin: 67 ± 4 (n = 54)].
Salivary gland degeneration assay There was a progressive 95% decline in salivary fluid secretory competence from day 0 post engorgement (PE; 4.60 ± 0.30 mg/gland/15 min) to day 4 PE (0.23 ± 0.06 mg/gland/15 min; Fig. 1) in the mated ticks. There was no significant change in the fluid secretory competence of the virgin tick SGs from day 0 PE (2.13 ± 0.12 mg/gland/15 min) to day 3 (1.8 ± 0.13 mg/gland/15 min). Rate of salivary fluid secretion of these glands did decrease substantially only after day 3 PE (p = 0.000, day 4 compared to day 3), and they eventually lost 90% of the initial fluid secretory competence by day 8 (0.12 ± 0.06 mg/gland/15 min; Fig. 1).
Ultrastructural evidence for salivary gland degeneration In ixodid ticks, the largest volume of saliva is secreted by the type III acini (Meredith and Kaufman, 1973). Figs. 2–6 show the ultrastructure of the fluid secretory labyrinth of type III acini of SGs as a function of days PE. Autophagic vacuoles, a strong indicator of tissue degeneration (Harris and Kaufman, 1981, 1984), were apparent already on day 0 PE (Fig. 2A and B). The apparent density of autophagic vacuoles increased progressively through day 4 (Figs. 3–5). The situation was the same for virgin ticks, except that
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Fig. 4. Fluid secretory type III acinus of (A) a 3-day PE-M (post engorgement-mated) D. andersoni female, 103× the unfed weight and (B) a 3-day PE-V (post engorgementvirgin) female, 83× the unfed weight. In both the mated and virgin female SGs, autophagic vacuoles are apparent. AV, autophagic vacuoles; L, salivary fluid secretory labyrinth.
they required 6–8 days to achieve a similar density of autophagic vacuoles that mated ticks achieved in 4 days (Fig. 6B).
Ovary development In the mated ticks, there was a progressive increase in ovary weight from day 0 through day 4 PE (1.1 ± 0.1% body weight to 10.1 ± 1.1% body weight; Fig. 7). The ovary weight then declined at day 6 (6.8 ± 0.5% body weight) perhaps because oviposition had begun by day 4 and was well underway by day 6. However, there was no further significant decline on day 8 (6.0 ± 1.5% body weight; p = 0.556 compared to day 6). On the other hand, there was no significant increase in ovary weight of the virgin ticks up to day 3 (3.9 ± 0.6% body weight to 4.7 ± 0.4% body weight; p = 0.772). Ovary weight then increased significantly on day 4 (9.4 ± 1.7% body weight; p = 0.002) and remained at this level thereafter up to day 8. In the majority of virgin ticks, oviposition had begun by day 8. In the mated ticks, oocyte length increased progressively from day 0 (188 ± 7 m) through day 4 PE (565 ± 29 m) and maintained this level up to day 8 (563 ± 8 m; p = 0.000, day 0 compared to day 4; Fig. 8). In the virgin ticks, there was no significant increase in oocyte length through day 3 (253 ± 23 m on day 0 and 284 ± 14 m on day 3; p = 0.141), but it increased on day 4
Fig. 5. Fluid secretory type III acinus of (A) a 4-day PE-M (post engorgement-mated) D. andersoni female, 80× the unfed weight and (B) a 4-day PE-V (post engorgementvirgin) female, 86× the unfed weight. In both the mated and virgin female SGs, the autophagic vacuoles are well-developed, and there appear to be fewer mitochondria than on earlier days. AV, autophagic vacuoles; L, salivary fluid secretory labyrinth; H, haemolymph.
(406 ± 18; p = 0.008, compared to day 3) and no further significant increase thereafter (Fig. 8). In mated ticks, the amount of vitellin increased steeply from day 0 (2.5 ± 0.2) through day 4 PE (196 ± 33; p = 0.000, day 4 compared to day 0; Fig. 9). For the virgin ticks, there was no significant change in vitellin content from day 0 (4.8 ± 0.6) through day 3 (7.8 ± 2.8; p = 0.288, day 3 compared to day 0), followed by a large increase on day 4 (96 ± 28), and a further increase on day 6 (173 ± 22) when the level plateaued (Fig. 9).
Discussion Since Till’s (1961) classic study there have been relatively few formal accounts focusing on SG degeneration in female ixodid ticks
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days post engorgement Fig. 7. Ovary weights of engorged mated and virgin D. andersoni ticks [expressed as % body weight (bw)] as a function of days post engorgement. Mean, SEM, and N are indicated for each bar. The increase of the ovary weight of the mated ticks was significant on day 1 (p = 0.000) compared to day 0. Ovary weight increase among the virgin ticks was significant only by day 4 (p = 0.018) compared to day 0.
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Fig. 6. Fluid secretory type III acinus of (A) a 6-day PE-V (post engorgement-virgin) D. andersoni female, 84× the unfed weight and (B) an 8-day PE-V female, 134× the unfed weight. The degree of degeneration appears similar in both panels. AV, autophagic vacuoles.
Fig. 8. Oocyte length of mated and virgin engorged D. andersoni ticks as a function of days post engorgement (PE). Mean, SEM, and N are indicated for each bar. In the mated ticks, a significant increase in the oocyte length was observed on day1 PE (p = 0.014) compared to day 0. In the virgin ticks, oocyte length increased significantly only by day 4 (p = 0.000) compared to day 0.
post engorgement. The most detailed contributions have been for A. hebraeum (reviewed by Kaufman et al., 1986). As mentioned in the ‘Introduction’, after feeding to repletion, tick salivary gland degeneration factor, an ecdysteroid hormone, triggers SG degeneration in A. hebraeum (Harris and Kaufman, 1981, 1985). But an additional factor, produced in the testes and transferred to the female during copulation, also plays a significant role in this process. This ‘male factor’, although not an essential mediator of SG degeneration (the SGs also degenerate in large virgin females, and ovarian development also proceeds to ovulation and oviposition), it hastens the process by 3–4 days (Lomas and Kaufman, 1992). The various delays that we observed in this study between virgin and mated D. andersoni suggest that a male factor is produced in this species as well. The cellular mechanisms accounting for salivary gland degeneration in ticks has commanded a fair amount of attention in the literature. Although it has long been assumed that SG degeneration is due to a highly regulated process like programmed cell
death/apoptosis rather than necrosis, direct evidence for this has been presented for several ticks in recent years. L’Amoreaux et al. (2003) monitored the time course of DNA fragmentation (an index of apoptosis) in the SGs of fully engorged female Dermacentor variabilis, using a TUNEL labelling assay. They observed no DNA fragmentation until 5 days PE, but apoptosis was observed thereafter through 33 days in the granular acini (types II and III), but not the agranular (type I) acini. Preservation of the type I acini is not only a general indicator that SG degeneration is a highly regulated rather than necrotic process, but is also consistent with the established role of the type I acini in water-balance physiology in off-host ticks (reviewed by Bowman et al., 2008). Freitas et al. (2007) conducted a study on programmed cell death in the SG, ovary, and synganglion of engorged female Rhipicephalus (Boophilus) microplus. Although the 3 distinct assays they used to monitor programmed cell death (TUNEL, comet and caspase-3) gave quantitatively distinct results, the general conclusion was that the process in all organs was well underway by 72 h PE, and that the SG (types II and III acini) was generally the most affected organ.
Vt content (400 500 nm per gram ovary)
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be acclimated to in the wild, and may contribute to why the onset of oviposition was earlier in D. andersoni in this study than has been reported for in A. hebraeum.
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days post engrogement Fig. 9. Ovary vitellin content in engorged (mated and virgin) D. andersoni ticks as a function of days post engorgement (PE). Vitellin content is expressed as the difference in absorbance between 400 and 500 nm per g ovary. Mean, SEM, and N are indicated for each bar. The increase of the vitellin content in the mated ticks from day 0 (2.5 ± 0.2) to day 4 PE (196 ± 33) was significant (p = 0.001, day 4 compared to day 0). Ovary vitellin content increased significantly in the virgin ticks only from day 4 (96 ± 28; p = 0.021) compared to day 0 (4.82 ± 0.61) and peaked at 173 ± 22 on day 6.
Furquim et al. (2008) likewise studied programmed cell death in the SGs of female Rhipicephalus sanguineus, comparing unfed, engorged day 0, and engorged day 3. Their study utilized cytochemical markers for acid phosphatase activity, RNA content, and ATPase activity in the cell membranes and cytoplasm in the 3 types of acini. In general, their results were consistent with those of L’Amoreaux et al. (2003) and Freitas et al. (2007), demonstrating weak or absent apoptotic activity in glands from unfed or day 0 PE ticks, and more intense apoptotic signals in glands from day 3 PE. Although the general conclusions of the latter 3 studies are consistent with each other, and with ours, there are significant differences with regard to the time course of onset and intensification of apoptotic signals. Species differences, of course, could be a factor. But the holding conditions of the ticks prior to dissection, may contribute to the differences as well. We, Freitas et al. (2007), and Furquim et al. (2008) maintained their ticks at 27 ◦ C, 28 ◦ C, and 29 ◦ C, respectively. But the SGs used by L’Amoreaux et al. (2003) were harvested from ticks that were held at a significantly lower 22 ◦ C (Lewis B. Coons, University of Memphis, personal communication, 10 February 2014). This may have contributed to a slower onset of apoptotic signals than occurred in the other studies. The rate of salivary fluid secretion in the day 0-mated engorged ticks was higher (by ∼50%; Fig. 1) than that of the virgin engorged ticks. This is not the case, however, in A. hebraeum, in which both mated and virgin ticks, above and below the so-called ‘critical weight’ secrete salivary fluid at a similar rate up to day 8 post removal (Harris and Kaufman, 1984). What accounts for this difference between the 2 species is not known. In this study, ovarian development occurred much more rapidly in D. andersoni than was reported earlier for A. hebraeum (e.g., Lunke and Kaufman, 1993; Friesen and Kaufman, 2002). Our colony incubator had been adjusted to be optimal for A. hebraeum (27 ◦ C); this is a somewhat higher average temperature that D. andersoni may
We thank Dr. Tym Lysyk (Agriculture and Agri-Food Canada, Lethbridge Research Centre, Canada) and Dr. Glen Scoles, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington and ARSUSDA, for providing us with the D. andersoni ticks used in this study. We also thank staff of the Biological Sciences Animal Facility and Arlene Oatway of the Biological Sciences Microscopy Unit, for their help with animal rearing and preparation of tissue samples for TEM respectively. This study was generously funded by a Discovery Grant 6479 to WRK from the Natural Sciences and Engineering Research Council (NSERC) of Canada. References Bowman, A.S., Ball, A., Sauer, J.R., 2008. Tick salivary glands: the physiology of tick water balance and their role in pathogen trafficking and transmission. In: Bowman, A.S., Nuttall, P.A. (Eds.), Ticks: Biology, Disease and Control. Cambridge University Press, Cambridge, England, UK, pp. 73–91. Coons, L.B., Kaufman, W.R., 1988. Evidence that developmental changes in type III acini in the tick Amblyomma hebraeum (Acari:Ixodidae) are initiated by a hemolymph-borne factor. Exp. Appl. Acarol. 4, 117–139. Freitas, D.R.J., Rosa, R.M., Moura, D.J., Seitz, A.L., Colodel, E.M., Driemeier, D., Da Silva Vaz Jr., I., Masuda, A., 2007. Cell death during preoviposition period in Boophilus microplus tick. Vet. Parasitol. 144, 321–327. Friesen, K.J., Kaufman, W.R., 2002. Quantification of vitellogenesis and its control by 20-hydroxyecdysone in the ixodid tick, Amblyomma hebraeum. J. Insect Physiol. 48, 773–782. Furquim, K.C.S., Bechara, G.H., Matias, M.I.C., 2008. Death by apoptosis in salivary glands of females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae). Exp. Parasitol. 119, 152–163. Harris, R.A., Kaufman, W.R., 1981. Hormonal control of salivary gland degeneration in the ixodid tick, Amblyomma hebraeum. J. Insect Physiol. 27, 241–248. Harris, R.A., Kaufman, W.R., 1984. Neural involvement in the control of salivary gland degeneration in the ixodid tick Amblyomma hebraeum. J. Exp. Biol. 109, 281– 290. Harris, R.A., Kaufman, W.R., 1985. Ecdysteroids: possible candidates for the hormone which triggers salivary gland degeneration in the ixodid tick Amblyomma hebraeum. Experientia 41, 740–742. Kaufman, W.R., 1976. The influence of various factors on fluid secretion in vitro salivary glands of ixodid ticks. J. Exp. Biol. 64, 727–742. Kaufman, W.R., 2007. Gluttony and sex in female ixodid ticks: how do they compare to other blood-sucking arthropods? J. Insect Physiol. 53, 264–273. Kaufman, W.R., Ungarian, S.G., Noga, A.E., 1986. The effect of avermectin on feeding, salivary fluid secretion and fecundity in some ticks. Exp. Appli. Acarol. 2, 1–18. L’Amoreaux, W.J., Junaid, L., Trevidi, S., 2003. Morphological evidence that salivary gland degeneration in the American dog tick, Dermacentor variabilis (Say), involves programmed cell death. Tissue Cell 35, 95–99. Lomas, L.O., Kaufman, W.R., 1992. The influence of a factor from the male genital tract on salivary gland degeneration in the female ixodid tick, Amblyomma hebraeum. J. Insect Physiol. 38, 595–601. Lunke, M.D., Kaufman, W.R., 1993. Hormonal control of ovarian development in the tick Amblyomma hebraeum Koch (Acari: Ixodidae). Invertebr. Reprod. Dev. 23, 25–38. Meredith, J., Kaufman, W.R., 1973. A proposed site of fluid secretion in the salivary gland of the ixodid tick Dermacentor andersoni. Parasitology 67, 205–217. Seixas, A., Friesen, K.J., Kaufman, W.R., 2008. Effect of 20-hydroxyecdysone and haemolymph on oogenesis in the ixodid tick Amblyomma hebraeum. J. Insect Physiol. 54, 1175–1183. Till, W.M., 1961. A contribution to the anatomy and histology of the brown ear tick, Rhipicephalus appendiculatus Neumann, 6. Memoirs of the Entomology Society of South Africa, 124 pp. Vitzthum, H.G., 1943. Acarina. In: Bronn’s Klassen and Ordnungen des Tierreiches, 5, IV. Abt., 5, Buch. Leipzig, Becker u. Erler.
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Ticks and Tick-borne Diseases journal homepage: www.elsevier.com/locate/ttbdis
Original article
Salivary gland degeneration and ovarian development in the Rocky Mountain wood tick, Dermacentor andersoni Stiles (Acari: Ixodidae). I. Post-engorgement events Shahid A.K.M. Ullah 1 , W. Reuben Kaufman ∗ Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6E 2E9
a r t i c l e
i n f o
Article history: Received 5 December 2013 Received in revised form 22 February 2014 Accepted 25 March 2014 Available online 24 June 2014 Keywords: Ixodid ticks Dermacentor andersoni Amblyomma hebraeum Salivary gland degeneration Ovary development Ovary vitellin content
a b s t r a c t It is well known that female ixodid ticks undergo salivary gland degeneration and the greater part of ovarian development following engorgement. The process has been particularly well studied in Amblyomma hebraeum, including the hormonal control of these processes. The purpose of this study, and the second one in this series, is to compare the processes in Dermacentor andersoni with those of A. hebraeum. A major difference between the two species is that virgin female D. andersoni feed to a much higher fed-tounfed weight ratio than do virgin female A. hebraeum, the former achieving an average of 67× the unfed weight in this study, the latter normally achieving an average of only about 10–15× the unfed weight. We show here that although engorged virgins (defined here as those that exceed a fed-unfed weight ratio of 50, even though they do not detach from the host spontaneously) degenerate their salivary glands and develop their ovaries, these processes proceed more slowly in virgins that they do in mated females, as is the case for A. hebraeum. © 2014 Elsevier GmbH. All rights reserved.
Introduction Female ixodid ticks, depending on species and stage of life cycle, require 5–10 days or so to engorge fully. During this period, the salivary glands (SGs) undergo an enormous degree of growth and development. For example, the maximum fluid secretory rate of isolated SGs from partially fed D. andersoni peaks at about 54× the rate observed for unfed ticks (Kaufman, 1976), a process that is controlled in A. hebraeum by a haemolymph-borne factor (Coons and Kaufman, 1988). However, within only about 4 days following engorgement, the SGs of A. hebraeum undergo autolysis; ovarian development, the early stages of which have begun during feeding, is completed within about 10–12 days post engorgement, when oviposition begins (Lunke and Kaufman, 1993; Friesen and Kaufman, 2002). The phenomenon of SG degeneration, based on histological data, has been noted since at least the 1940s (Vitzthum, 1943, quoted by Till, 1961), and subsequently well established by the 1960s (Till, 1961). The hormonal control of SG degeneration was
first demonstrated by Harris and Kaufman (1981) in A. hebraeum; they named the hormone ‘tick salivary gland degeneration factor’. A few years later, tick salivary gland degeneration factor was shown to be an ecdysteroid hormone (Harris and Kaufman, 1985). Like many ixodid ticks, A. hebraeum must copulate before they can engorge. The approximate maximum fed-to-unfed weight ratio achieved by most virgin A. hebraeum is only about 10, a ratio that is too low to trigger SG degeneration or ovarian development (Kaufman, 2007). But virgin D. andersoni normally feed to a much higher weight ratio than do A. hebraeum (Kaufman, 2007). Even though such large virgins do not usually detach spontaneously from the host, they are at a size sufficient to trigger SG degeneration and ovarian development. The objective of this study was to determine the dynamics of SG degeneration and ovarian development in normally engorged mated D. andersoni and in nominally “engorged” virgins (those that achieve >50× the unfed weight, even though they do not detach spontaneously), and to compare these dynamics with those previously established for A. hebraeum. Materials and methods
∗ Corresponding author at: Cormorant Crescent, Salt Spring Island, BC, Canada V8K 1G8. Tel.: +1 250 931 0033. E-mail address: [email protected] (W.R. Kaufman). 1 Present address: Unit 101, 10710, 85 Avenue NW, Edmonton, Alberta, Canada T6E 2K8. http://dx.doi.org/10.1016/j.ttbdis.2014.03.012 1877-959X/© 2014 Elsevier GmbH. All rights reserved.
Ticks The adult D. andersoni ticks used in this study were provided by Dr. Tym Lysyk (Agriculture and Agri-Food Canada, Lethbridge Research Centre, Canada), mean female unfed weight of
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6.47 ± 0.13 mg (n = 260), and by Dr. Glen Scoles, (Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington and ARS, USDA), mean female unfed weight of 4.67 ± 0.06 mg (n = 328). As we frequently compare the results of this study with those done previously using A. hebraeum, the mean unfed weight for a large sample of the A. hebraeum females from our recent colony was 30.0 ± 0.6 mg (n = 192). After arriving at our laboratory, ticks were maintained in darkness, at 7–9 ◦ C, and held over saturated KNO3 (RH ∼95%). The day before putting them on a New Zealand white rabbit (SPF; Charles River, Senneville, Quebec, Canada), the ticks were transferred to a humid, 27 ◦ C incubator. A cloth-covered foam ‘backpack’ (∼12 cm × 8 cm × 2.5 cm) was glued to the shaven back of the rabbit with a latex adhesive (Roberts 8502 Latex, Bramalea, Ontario, Canada). In order to determine the ultimate fed-to-unfed weight ratio of each experimental tick, the ticks were coded prior to feeding by glueing a coloured silk thread to a leg, and then weighed. Equal numbers of males and females were introduced together to the backpack for experiments requiring mated females; only females were added to the backpack for experiments requiring virgin females. Mated females normally engorged and detached spontaneously after 4–6 days of feeding. Virgin females that were allowed to feed on the host for 21 days (the maximum allowed by our animal use protocol) and which exceeded about 50× the unfed weight, were defined as “fully engorged”, even though they did not detach from the host spontaneously. After collecting the ticks from the host, they were rinsed with water, dried with tissue paper, weighed, and stored (27 ◦ C) individually in cloth-covered plastic vials until dissection. Our animal-use protocol was approved by the Biosciences Animal Policy and Welfare Committee of the University of Alberta. This committee functions according to the current guidelines established by the Canadian Council on Animal Care. Assay for salivary fluid secretory competence The technique used was based on that described by Harris and Kaufman (1984). Briefly, ticks were glued ventral surface down to a disposable Petri dish and cooled in a refrigerator for about 30 min. They were then bathed in modified Hank’s saline (per litre: NaCl 11.5 g, d-glucose 1.6 g, KCl 0.4 g, phenol red 0.01 g; the pH was adjusted to 7.2) and the SGs and ovary dissected out. The tissues were transferred to TC medium 199 made with Hank’s salts (Sigma Chemical Company, St. Louise, MO, USA). Before dissolving a package of the powdered medium in 1 L milliQ (Millipore) water, we added 2.09 g of the buffer, morpholinopropanesulphonic acid (MOPS), plus 2.1 g NaCl; the pH was adjusted to 7.2. The SGs and ovary were dissected free of extraneous tissues. The main salivary duct was ligated with a fine strand peeled from silk thread. Each gland was transferred to a hydrophobic surface and extraglandular fluid was removed by gentle application of a piece of filter paper. The gland was then weighed to the nearest 10 g on a 5-place electronic balance and immediately transferred to a bathing medium of TC medium 199 containing freshly prepared dopamine (10 M; Sigma Chemical Co.). The bathing medium was slowly agitated with a magnetic stir-bar during the 15-min incubation period. The increase in wet weight per gland between 0 and 15 min was used as an index of salivary fluid secretory competence (Harris and Kaufman, 1984). Dopamine is probably the natural neurotransmitter for salivary fluid secretion, and 10 M is a supra-maximal concentration (Kaufman, 1976). Ovary maturation assays The ovary was dissected out and submerged in TC medium 199, and the lengths of 10 apparently largest oocytes were measured
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days post engorgement Fig. 1. Rate of salivary fluid uptake in engorged (mated and virgin) D. andersoni females as a function of days post engorgement. Mean, SEM, and N are indicated for each bar. Among the mated ticks, a significant loss of fluid secretory competence occurred throughout 4 days post engorgement (in all cases p = 0.000 in comparison to day 0). Among the virgin ticks, a significant loss of fluid secretory competence began only on day 4 post engorgement (p = 0.000 in comparison to day 0).
using a dissecting microscope fitted with an ocular micrometre. The ovary was gently blotted with filter paper and weighed to the nearest 10 g. The ovary was then homogenized in 1 ml milliQ water, frozen quickly on dry ice, and stored at −20 ◦ C in small polyethylene microfuge tubes. For the spectrophotometric analysis of vitellin content, ovary homogenates were thawed and diluted with milliQ water to a final volume of 2 ml. After gentle vortexing, the homogenates were centrifuged at 12,000 rpm for 10 min. The supernatant was collected and stored at −20 ◦ C. For the final analysis, samples and blank were thawed, and the absorbance at 400 nm (specific for the hem moiety of vitellin) and 500 nm (non-specific for hem) were measured on a spectrophotometer (Ultrospec 3300 pro, Biochrom Ltd, Cambridge, England). The difference between the 2 absorbance readings was accepted as a measure of vitellin content of the sample and was normalized to ovary weight (expressed as 400 nm 500 nm per gram ovary; Kaufman et al., 1986; Seixas et al., 2008). Transmission electron microscopy (TEM) of salivary glands The technique was based on the optimized protocol used in the Microscopy Unit, Department of Biological Sciences. Briefly, SGs were dissected out under Hank’s saline. The large tracheae were dissected away, and the glands were fixed at 4 ◦ C for at least 2 h in a solution containing 2.5% glutaraldehyde, 2% paraformaldehyde, 0.1 M phosphate buffer (pH 7.3). After fixation, the tissue was washed 4 times with 0.1 M phosphate buffer (2 quick changes followed by 2 changes 10 min apart). The glands were post-fixed with 1% OsO4 in 0.12 M cacodylate buffer, pH 7.2. The glands were washed again with 3 changes of 0.1 M phosphate buffer over 30–60 min. The glands were then dehydrated through a graded ethanol series (50%, 70%, 90%, 15 min each change), followed by 3 changes of absolute ethanol. The glands were incubated overnight in 1:1 ethanol:low viscosity spur resin (Electron Microscopy Sciences, Hatfield, PA, USA). This was followed by 2 changes of pure spur resin, a few hours apart. The glands in resin were then transferred to moulds and held at 70–80 ◦ C for 16–18 h. Tissue blocks were sectioned (70–90 nm thick) using an Ultracut E Reichert-Jung Ultramicrotome. Sections were transferred to copper grids (200 mesh). The sections were stained with 4% uranyl acetate for 25 min and counterstained in lead citrate (per 50 ml solution: 1.33 g lead
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Fig. 3. Fluid secretory type III acinus of (A) a 2-day PE-M (post engorgementmated) D. andersoni female; 96× the unfed weight and (B) a 2-day PE-V (post engorgement-virgin) female; 102× the unfed weight. In both mated and virgin female SGs, autophagic vacuoles are apparent, although they are still more prominent in the mated female SG. AV, autophagic vacuoles; M, mitochondria; L, salivary fluid secretory labyrinth. Fig. 2. Fluid secretory type III acinus of (A) a 0-day PE-M (post engorgementmated) D. andersoni female; 93× the unfed weight and (B) a 0-day PE-V (post engorgement-virgin) female, 81× the unfed weight. In both mated and virgin female SGs, autophagic vacuoles are apparent, although they appear to be more prominent in the mated female SG. AV, autophagic vacuoles; M, mitochondria; L, salivary fluid secretory labyrinth; H, haemolymph.
citrate, 1.76 g sodium citrate in double distilled, vacuum-filtered H2 O, pH-adjusted to 12.0 by 0.01 N NaOH) for 2 min. Grids were examined by TEM (Philips Morgagni 268 and Philips 410, operating at 80 kV), and photomicrographs were taken with a Gatan Orius CCD camera. Statistics Unless otherwise stated, all data are reported as mean ± SEM (N), using Microsoft Excel software (Microsoft Office 2007). Statistical significance was determined by student’s t-test using STATA 10.0 (StataCorp, TX, USA) on a Macintosh Computer. When analyzing the effect of time post engorgement, the comparator group was always day 0. Other comparisons will be specified within figure legends. Results The fed/unfed weight ratio for engorged D. andersoni females used in this experiment was in the range of 50–100 [average for mated: 87 ± 7 (n = 62); average for virgin: 67 ± 4 (n = 54)].
Salivary gland degeneration assay There was a progressive 95% decline in salivary fluid secretory competence from day 0 post engorgement (PE; 4.60 ± 0.30 mg/gland/15 min) to day 4 PE (0.23 ± 0.06 mg/gland/15 min; Fig. 1) in the mated ticks. There was no significant change in the fluid secretory competence of the virgin tick SGs from day 0 PE (2.13 ± 0.12 mg/gland/15 min) to day 3 (1.8 ± 0.13 mg/gland/15 min). Rate of salivary fluid secretion of these glands did decrease substantially only after day 3 PE (p = 0.000, day 4 compared to day 3), and they eventually lost 90% of the initial fluid secretory competence by day 8 (0.12 ± 0.06 mg/gland/15 min; Fig. 1).
Ultrastructural evidence for salivary gland degeneration In ixodid ticks, the largest volume of saliva is secreted by the type III acini (Meredith and Kaufman, 1973). Figs. 2–6 show the ultrastructure of the fluid secretory labyrinth of type III acini of SGs as a function of days PE. Autophagic vacuoles, a strong indicator of tissue degeneration (Harris and Kaufman, 1981, 1984), were apparent already on day 0 PE (Fig. 2A and B). The apparent density of autophagic vacuoles increased progressively through day 4 (Figs. 3–5). The situation was the same for virgin ticks, except that
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Fig. 4. Fluid secretory type III acinus of (A) a 3-day PE-M (post engorgement-mated) D. andersoni female, 103× the unfed weight and (B) a 3-day PE-V (post engorgementvirgin) female, 83× the unfed weight. In both the mated and virgin female SGs, autophagic vacuoles are apparent. AV, autophagic vacuoles; L, salivary fluid secretory labyrinth.
they required 6–8 days to achieve a similar density of autophagic vacuoles that mated ticks achieved in 4 days (Fig. 6B).
Ovary development In the mated ticks, there was a progressive increase in ovary weight from day 0 through day 4 PE (1.1 ± 0.1% body weight to 10.1 ± 1.1% body weight; Fig. 7). The ovary weight then declined at day 6 (6.8 ± 0.5% body weight) perhaps because oviposition had begun by day 4 and was well underway by day 6. However, there was no further significant decline on day 8 (6.0 ± 1.5% body weight; p = 0.556 compared to day 6). On the other hand, there was no significant increase in ovary weight of the virgin ticks up to day 3 (3.9 ± 0.6% body weight to 4.7 ± 0.4% body weight; p = 0.772). Ovary weight then increased significantly on day 4 (9.4 ± 1.7% body weight; p = 0.002) and remained at this level thereafter up to day 8. In the majority of virgin ticks, oviposition had begun by day 8. In the mated ticks, oocyte length increased progressively from day 0 (188 ± 7 m) through day 4 PE (565 ± 29 m) and maintained this level up to day 8 (563 ± 8 m; p = 0.000, day 0 compared to day 4; Fig. 8). In the virgin ticks, there was no significant increase in oocyte length through day 3 (253 ± 23 m on day 0 and 284 ± 14 m on day 3; p = 0.141), but it increased on day 4
Fig. 5. Fluid secretory type III acinus of (A) a 4-day PE-M (post engorgement-mated) D. andersoni female, 80× the unfed weight and (B) a 4-day PE-V (post engorgementvirgin) female, 86× the unfed weight. In both the mated and virgin female SGs, the autophagic vacuoles are well-developed, and there appear to be fewer mitochondria than on earlier days. AV, autophagic vacuoles; L, salivary fluid secretory labyrinth; H, haemolymph.
(406 ± 18; p = 0.008, compared to day 3) and no further significant increase thereafter (Fig. 8). In mated ticks, the amount of vitellin increased steeply from day 0 (2.5 ± 0.2) through day 4 PE (196 ± 33; p = 0.000, day 4 compared to day 0; Fig. 9). For the virgin ticks, there was no significant change in vitellin content from day 0 (4.8 ± 0.6) through day 3 (7.8 ± 2.8; p = 0.288, day 3 compared to day 0), followed by a large increase on day 4 (96 ± 28), and a further increase on day 6 (173 ± 22) when the level plateaued (Fig. 9).
Discussion Since Till’s (1961) classic study there have been relatively few formal accounts focusing on SG degeneration in female ixodid ticks
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days post engorgement Fig. 7. Ovary weights of engorged mated and virgin D. andersoni ticks [expressed as % body weight (bw)] as a function of days post engorgement. Mean, SEM, and N are indicated for each bar. The increase of the ovary weight of the mated ticks was significant on day 1 (p = 0.000) compared to day 0. Ovary weight increase among the virgin ticks was significant only by day 4 (p = 0.018) compared to day 0.
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Fig. 6. Fluid secretory type III acinus of (A) a 6-day PE-V (post engorgement-virgin) D. andersoni female, 84× the unfed weight and (B) an 8-day PE-V female, 134× the unfed weight. The degree of degeneration appears similar in both panels. AV, autophagic vacuoles.
Fig. 8. Oocyte length of mated and virgin engorged D. andersoni ticks as a function of days post engorgement (PE). Mean, SEM, and N are indicated for each bar. In the mated ticks, a significant increase in the oocyte length was observed on day1 PE (p = 0.014) compared to day 0. In the virgin ticks, oocyte length increased significantly only by day 4 (p = 0.000) compared to day 0.
post engorgement. The most detailed contributions have been for A. hebraeum (reviewed by Kaufman et al., 1986). As mentioned in the ‘Introduction’, after feeding to repletion, tick salivary gland degeneration factor, an ecdysteroid hormone, triggers SG degeneration in A. hebraeum (Harris and Kaufman, 1981, 1985). But an additional factor, produced in the testes and transferred to the female during copulation, also plays a significant role in this process. This ‘male factor’, although not an essential mediator of SG degeneration (the SGs also degenerate in large virgin females, and ovarian development also proceeds to ovulation and oviposition), it hastens the process by 3–4 days (Lomas and Kaufman, 1992). The various delays that we observed in this study between virgin and mated D. andersoni suggest that a male factor is produced in this species as well. The cellular mechanisms accounting for salivary gland degeneration in ticks has commanded a fair amount of attention in the literature. Although it has long been assumed that SG degeneration is due to a highly regulated process like programmed cell
death/apoptosis rather than necrosis, direct evidence for this has been presented for several ticks in recent years. L’Amoreaux et al. (2003) monitored the time course of DNA fragmentation (an index of apoptosis) in the SGs of fully engorged female Dermacentor variabilis, using a TUNEL labelling assay. They observed no DNA fragmentation until 5 days PE, but apoptosis was observed thereafter through 33 days in the granular acini (types II and III), but not the agranular (type I) acini. Preservation of the type I acini is not only a general indicator that SG degeneration is a highly regulated rather than necrotic process, but is also consistent with the established role of the type I acini in water-balance physiology in off-host ticks (reviewed by Bowman et al., 2008). Freitas et al. (2007) conducted a study on programmed cell death in the SG, ovary, and synganglion of engorged female Rhipicephalus (Boophilus) microplus. Although the 3 distinct assays they used to monitor programmed cell death (TUNEL, comet and caspase-3) gave quantitatively distinct results, the general conclusion was that the process in all organs was well underway by 72 h PE, and that the SG (types II and III acini) was generally the most affected organ.
Vt content (400 500 nm per gram ovary)
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be acclimated to in the wild, and may contribute to why the onset of oviposition was earlier in D. andersoni in this study than has been reported for in A. hebraeum.
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days post engrogement Fig. 9. Ovary vitellin content in engorged (mated and virgin) D. andersoni ticks as a function of days post engorgement (PE). Vitellin content is expressed as the difference in absorbance between 400 and 500 nm per g ovary. Mean, SEM, and N are indicated for each bar. The increase of the vitellin content in the mated ticks from day 0 (2.5 ± 0.2) to day 4 PE (196 ± 33) was significant (p = 0.001, day 4 compared to day 0). Ovary vitellin content increased significantly in the virgin ticks only from day 4 (96 ± 28; p = 0.021) compared to day 0 (4.82 ± 0.61) and peaked at 173 ± 22 on day 6.
Furquim et al. (2008) likewise studied programmed cell death in the SGs of female Rhipicephalus sanguineus, comparing unfed, engorged day 0, and engorged day 3. Their study utilized cytochemical markers for acid phosphatase activity, RNA content, and ATPase activity in the cell membranes and cytoplasm in the 3 types of acini. In general, their results were consistent with those of L’Amoreaux et al. (2003) and Freitas et al. (2007), demonstrating weak or absent apoptotic activity in glands from unfed or day 0 PE ticks, and more intense apoptotic signals in glands from day 3 PE. Although the general conclusions of the latter 3 studies are consistent with each other, and with ours, there are significant differences with regard to the time course of onset and intensification of apoptotic signals. Species differences, of course, could be a factor. But the holding conditions of the ticks prior to dissection, may contribute to the differences as well. We, Freitas et al. (2007), and Furquim et al. (2008) maintained their ticks at 27 ◦ C, 28 ◦ C, and 29 ◦ C, respectively. But the SGs used by L’Amoreaux et al. (2003) were harvested from ticks that were held at a significantly lower 22 ◦ C (Lewis B. Coons, University of Memphis, personal communication, 10 February 2014). This may have contributed to a slower onset of apoptotic signals than occurred in the other studies. The rate of salivary fluid secretion in the day 0-mated engorged ticks was higher (by ∼50%; Fig. 1) than that of the virgin engorged ticks. This is not the case, however, in A. hebraeum, in which both mated and virgin ticks, above and below the so-called ‘critical weight’ secrete salivary fluid at a similar rate up to day 8 post removal (Harris and Kaufman, 1984). What accounts for this difference between the 2 species is not known. In this study, ovarian development occurred much more rapidly in D. andersoni than was reported earlier for A. hebraeum (e.g., Lunke and Kaufman, 1993; Friesen and Kaufman, 2002). Our colony incubator had been adjusted to be optimal for A. hebraeum (27 ◦ C); this is a somewhat higher average temperature that D. andersoni may
We thank Dr. Tym Lysyk (Agriculture and Agri-Food Canada, Lethbridge Research Centre, Canada) and Dr. Glen Scoles, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington and ARSUSDA, for providing us with the D. andersoni ticks used in this study. We also thank staff of the Biological Sciences Animal Facility and Arlene Oatway of the Biological Sciences Microscopy Unit, for their help with animal rearing and preparation of tissue samples for TEM respectively. This study was generously funded by a Discovery Grant 6479 to WRK from the Natural Sciences and Engineering Research Council (NSERC) of Canada. References Bowman, A.S., Ball, A., Sauer, J.R., 2008. Tick salivary glands: the physiology of tick water balance and their role in pathogen trafficking and transmission. In: Bowman, A.S., Nuttall, P.A. (Eds.), Ticks: Biology, Disease and Control. Cambridge University Press, Cambridge, England, UK, pp. 73–91. Coons, L.B., Kaufman, W.R., 1988. Evidence that developmental changes in type III acini in the tick Amblyomma hebraeum (Acari:Ixodidae) are initiated by a hemolymph-borne factor. Exp. Appl. Acarol. 4, 117–139. Freitas, D.R.J., Rosa, R.M., Moura, D.J., Seitz, A.L., Colodel, E.M., Driemeier, D., Da Silva Vaz Jr., I., Masuda, A., 2007. Cell death during preoviposition period in Boophilus microplus tick. Vet. Parasitol. 144, 321–327. Friesen, K.J., Kaufman, W.R., 2002. Quantification of vitellogenesis and its control by 20-hydroxyecdysone in the ixodid tick, Amblyomma hebraeum. J. Insect Physiol. 48, 773–782. Furquim, K.C.S., Bechara, G.H., Matias, M.I.C., 2008. Death by apoptosis in salivary glands of females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae). Exp. Parasitol. 119, 152–163. Harris, R.A., Kaufman, W.R., 1981. Hormonal control of salivary gland degeneration in the ixodid tick, Amblyomma hebraeum. J. Insect Physiol. 27, 241–248. Harris, R.A., Kaufman, W.R., 1984. Neural involvement in the control of salivary gland degeneration in the ixodid tick Amblyomma hebraeum. J. Exp. Biol. 109, 281– 290. Harris, R.A., Kaufman, W.R., 1985. Ecdysteroids: possible candidates for the hormone which triggers salivary gland degeneration in the ixodid tick Amblyomma hebraeum. Experientia 41, 740–742. Kaufman, W.R., 1976. The influence of various factors on fluid secretion in vitro salivary glands of ixodid ticks. J. Exp. Biol. 64, 727–742. Kaufman, W.R., 2007. Gluttony and sex in female ixodid ticks: how do they compare to other blood-sucking arthropods? J. Insect Physiol. 53, 264–273. Kaufman, W.R., Ungarian, S.G., Noga, A.E., 1986. The effect of avermectin on feeding, salivary fluid secretion and fecundity in some ticks. Exp. Appli. Acarol. 2, 1–18. L’Amoreaux, W.J., Junaid, L., Trevidi, S., 2003. Morphological evidence that salivary gland degeneration in the American dog tick, Dermacentor variabilis (Say), involves programmed cell death. Tissue Cell 35, 95–99. Lomas, L.O., Kaufman, W.R., 1992. The influence of a factor from the male genital tract on salivary gland degeneration in the female ixodid tick, Amblyomma hebraeum. J. Insect Physiol. 38, 595–601. Lunke, M.D., Kaufman, W.R., 1993. Hormonal control of ovarian development in the tick Amblyomma hebraeum Koch (Acari: Ixodidae). Invertebr. Reprod. Dev. 23, 25–38. Meredith, J., Kaufman, W.R., 1973. A proposed site of fluid secretion in the salivary gland of the ixodid tick Dermacentor andersoni. Parasitology 67, 205–217. Seixas, A., Friesen, K.J., Kaufman, W.R., 2008. Effect of 20-hydroxyecdysone and haemolymph on oogenesis in the ixodid tick Amblyomma hebraeum. J. Insect Physiol. 54, 1175–1183. Till, W.M., 1961. A contribution to the anatomy and histology of the brown ear tick, Rhipicephalus appendiculatus Neumann, 6. Memoirs of the Entomology Society of South Africa, 124 pp. Vitzthum, H.G., 1943. Acarina. In: Bronn’s Klassen and Ordnungen des Tierreiches, 5, IV. Abt., 5, Buch. Leipzig, Becker u. Erler.
