Morphological evidence that salivary gland degeneration in the American dog tick, Dermacentor variabilis (Say), involves programmed cell death

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1 Tissue & Cell 35 (2003) Morphological evidence that salivary gland degeneration in the American dog tick, Dermacentor variabilis (Say), involves programmed cell death W.J. L Amoreaux, L. Junaid, S. Trevidi Department of Biology, College of Staten Island, The City University of New York, 2800 Victory Blvd., Staten Island, NY 10314, USA Received 4 November 2002; received in revised form 9 December 2002; accepted 9 December 2002 Abstract During the preoviposition and oviposition periods of ixodid ticks, the salivary glands degenerate. It is unclear whether this is a necrotic or a programmed cell death event. We used an in situ TUNEL technique to determine if salivary gland degeneration involves apoptosis. Salivary glands were dissected from replete females at days 3, 5, 8, 11, 13, and 33 post-detachment. There were no differences in tick weight at detachment, suggesting that changes were not due to engorgement abnormalities. The onset of apoptosis began at day 5 and continued through oviposition at day 33. The greatest amount of nuclei containing fragmented DNA was observed on day 8 post-detachment, suggesting this was the peak occurrence of programmed cell death. Further, the temporal organization of programmed cell death suggests that the granule-secreting acini undergo apoptosis first, and that during the first week of oviposition the type I acini do not exhibit programmed cell death. These data suggest that the type I acini may still function in maintaining off-host hydration state of ovipositing females. These data provide morphological evidence that salivary gland degeneration involves a temporal programmed cell death event Elsevier Science Ltd. All rights reserved. Keywords: Apoptosis; Ixodidae; Salivary glands; Ticks; Acari 1. Introduction Ixodid ticks are obligate bloodsucking arthropods that are vectors of several viral and bacterial diseases, including Lyme disease, Rocky Mountain spotted fever, erhlichiosis, and theileriosis. The pathogens are transmitted from the vector to the host by way of the salivary glands. In addition, the salivary glands are responsible for secretion of the attachment cement that allows the parasite to remain attached to the host for upwards of 14 days. During the time on-host, the tick is required to evade the host s immune system and does so through salivary gland secretions. The salivary glands, therefore, are of great interest for on-host activities. The salivary glands of ixodid ticks are paired structures that occupy approximately two thirds of the anterior portion of the hemocoel. These organs are comprised of three types of acini (types I, II, and III) in the female. The acini are, in turn, comprised of several cell types (Till, 1959). Type I acini are agranular and are located nearest to the mouthparts (Fawcett et al., 1986). The function of the type I acini in unfed ticks is to main the hydration state of the tick while off-host (Needham et al., 1990). The types II and III acini in Corresponding author. Tel.: ; fax: address: lamoreaux@mail.csi.cuny.edu (W.J. L Amoreaux). the female contain granule-secreting cells as well as ablumenal and adlumenal interstitial cells (Fawcett et al., 1981; Till, 1959). The ablumenal cell in both acini type takes on an ultrastructure characteristic of fluid-transporting cells (Coons and Lamoreaux, 1986). The type III acini are responsible for osmoregulation while the tick is feeding, with excess water and ions from the blood meal returned to the host during salivation (Tatchell, 1967, 1969). Fluid is taken up from the hemolymph and is transported into the type III acini via the ablumenal cells. The fluid is subsequently expelled into the salivary ducts by the adlumenal cell (Coons et al., 1994; Lamoreaux et al., 1994). The adlumenal interstitial cell is a myoepithelial cells that requires an intact actin network for contractions and subsequent salivation to occur (Coons et al., 1994; Lamoreaux et al., 1994). There is only one adlumenal cell per type III acinus and the cells apical border adjoins the lumen of the acinus (Coons and Lamoreaux, 1986). During on-host feeding activities, the salivary glands undergo significant biochemical and morphological changes in order to be secretory competent. The biochemical and physiological changes involved in the development of the salivary glands have recently been reviewed elsewhere (Sauer et al., 2000). Of the three types of acini in the female, the types II and III undergo morphological changes, with type III displaying the most dramatic changes (Coons and Lamoreaux, /03/$ see front matter 2003 Elsevier Science Ltd. All rights reserved. doi: /s (02)00109-x

2 96 W.J. L Amoreaux et al. / Tissue & Cell 35 (2003) ). In the type II acinus, the adlumenal and ablumenal interstitial cells show an increase in size, with the ablumenal interstitial cell showing the greater change of the two. In the type III acinus, both interstitial cells increase in size, but it is the ablumenal cell that shows the greatest morphological changes (Coons and Lamoreaux, 1986). The type III acinus also shows a feeding-related change in the f-cell, beginning with the accumulation and secretion of electron-dense granules during the first 72 h of feeding. These changes are followed by a re-organization of the rough endoplasmic reticulum and the elaboration of Golgi-derived vacuole-like structures during the rapid engorgement-feeding period (Coons and Lamoreaux, 1986). Once the ixodid female is replete and detaches from the vertebrate host, the salivary glands are no longer needed. The female will use the blood meal to convert to vitellogenin for incorporation into the yolk as the yolk protein vitellin (Coons et al., 1989; Tarnowski and Coons, 1989). Upon reaching the replete weight, the salivary glands of the female Amblyomma hebraeum begin to degenerate (Harris and Kaufman, 1981). The ultrastructure observed in the type III acini of degenerating salivary glands is reminiscent of the development of lysosomal vacuoles observed in the labial gland of Manduca sexta and salivary glands of Drosophila (Zakeri, 1998). In this tick, salivary gland degeneration is almost complete within 4 days following detachment from the host. It is interesting to note that in Dermacentor variabilis, salivary gland remnants remain through at least day 33 post-detachment. This suggests that within the Ixodidae, salivary gland degeneration may proceed at greatly different rates. The onset of salivary gland degeneration coincides with an increase in the levels of 20-hydroxyecdysone in the hemolymph (Harris and Kaufman, 1985; Mao and Kaufman, 1999). Ecdysteroids have been determined to induce apoptosis in intersegmental muscle, prothoracic gland, and accessory plant retractor motoneurons of Manduca (Dai and Gilbert, 1999; Hoffman and Weeks, 1998; Kuelzer et al., 1999) as well as larval brains of Sarcophaga peregrina (Fujii et al., 1999). Together, the data suggest that the mechanisms underlying salivary gland degeneration in ixodid ticks is programmed in response to hormonal stimulation of an ecdysteroid receptor. The purpose of this study was to examine the salivary glands of replete D. variabilis to determine if there are morphological changes in the three acini characteristic of programmed cell death. 2. Materials and methods 2.1. Ticks Adult female D. variabilis were fed in the presence of male ticks on New Zealand White rabbits or Suffolk sheep as previously described (Patrick and Hair, 1975) under approved Institutional Animal Care and Use Committee protocols. Replete females were collected, immobilized in paraffin, and dissected under 4% paraformaldehyde. Glands were removed from the replete females on days 3, 5, 8, 11, 13, and 33 post-detachment under fixative and removed to fresh fixative, where the glands were stored at 4 C until analysis Apoptosis detection DNA fragmentation, a characteristic nuclear event during apoptosis, was determined in situ by incorporating labeled nucleotides onto the free 3 -hydroxyl ends using terminal deoxynucleotidyl transferase (TdT) and digoxigenin-labeled nucleotide triphosphates. A commercially available kit was used that employed fluorescence detection of labeled cells (Intergen). The labeled nucleotides were detected using primary antibodies directed against digoxigenin-labeled dutp. Primary antibodies directed against digoxigenin were detected with FITC-labeled secondary antibodies. Cells were counterstained with propidium iodide, which is excluded from nuclei that have begun apoptosis (Ankarcrona et al., 1995). 3. Results We have used the TUNEL labeling technique in conjunction with immunohistochemical detection of digoxigeninlabeled dutp to demonstrate DNA fragmentation in salivary glands of replete D. variabilis females. We have examined the salivary glands of females who have been Fig. 1. Fluorescence micrographs of salivary glands of replete female Dermacentor variabilis. The glands were removed as described in the Section 2 and incubated with digoxigenin-labeled dutp and terminal deoxynucleotidyl transferase (TdT) in a modified TUNEL technique. Intact, non-apoptotic nuclei were labeled with propidium iodide (PI). (A) Glands from replete female 3 days following detachment. The PI-labeled nuclei (Nu) remain intact and show no signs of apoptosis. Secretory granules (G) were observed as autofluorescence as were the chitinous lining of the salivary duct (SD). (B) Glands from replete female 5 days following detachment. Numerous apoptotic nuclei were observed (arrows) as were intact non-apoptotic nuclei (Nu). (C) Glands from replete female 8 days following detachment. Numerous apoptotic nuclei were observed (arrows) as were intact non-apoptotic nuclei (Nu). Note the apoptotic nuclei have a distinct labeling size as compared to the autofluorescent granules in panel A. (D) Glands from replete female 11 days following detachment. Fewer apoptotic nuclei were observed (arrows) and the number of intact non-apoptotic nuclei (Nu) remained relatively constant. Most of the intact nuclei are from type I acini. (E) Glands from replete female 13 days following detachment. The number of apoptotic events were not significantly different from day 11. This micrograph shows an isolated granule-secreting acinus (granules were below the focal plane). (F) Glands from replete female 33 days following detachment. Scattered apoptotic nuclei were observed (arrows). Most of the non-apoptotic nuclei (Nu) appeared along the secretory duct and in what appears to be type I acini.

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4 98 W.J. L Amoreaux et al. / Tissue & Cell 35 (2003) off-host for 3, 5, 8, 11, 13, and 33 days. In the third day following detachment, there was no indication that fragmentation had occurred (Fig. 1A). This may be the result of no apoptotic activity, but may be due to the masking effect of aldehyde fixation for a period greater than 3 weeks (Labat-Moleur et al., 1998). We then examined glands from replete females that had been detached for 5 days, a time in which apoptotic events should be well established. A number of apoptotic cells were present and could be distinguished from autofluorescent granules of the types II and III acini (Fig. 1B). In particular, the type III acini appear to be the most affected by DNA fragmentation, suggesting that a temporal organization exists among the acini types. Apoptosis continued through days 8, 11, and 13, although the later stage glands had suffered significant morphological disruption (Fig. 1C E). In particular, we see more type II acini, yet no type I acini, are undergoing programmed cell death by days By day 33, most of the glands had degenerated. The only nuclei observed were the epithelial cells of the duct and a few apoptotic nuclei of presumably type I acini (Fig. 1F). 4. Discussion We provide here morphological data that suggests that salivary gland degeneration involves, at least in part, type I programmed cell death. We examined the degree of apoptosis as indicated by DNA fragmentation from preoviposition through oviposition. In particular, we began our examinations on the glands from females that had been off-host for 3 or more days. The rationale for these times arise from observations that secretory competence in the ixodid ticks A. hebraeum and Amblyomma americanum begins to decline at days 3 4 post-detachment (Harris and Kaufman, 1984; Lindsay and Kaufman, 1988). This loss of secretory competence precedes the onset of degeneration. Salivary gland degeneration continues through day 5 post-detachment and coincides with rises in hemolymph ecdysteroid titers (Kaufman, 1991). Our morphological data support Kaufman s observations that salivary gland degeneration does begin through day 5 in Amblyomma. Early investigations into ixodid salivary gland physiology suggest that there are little or no differences between Amblyomma and Dermacentor species in the timing of degeneration (Kaufman, 1976). We would, therefore, expect that many of the initial events are similar in both genera. Further, we show in this study that actual degeneration of glands continues through day 33, a time in which oviposition is ending. Because of the volume of cells comprising the total salivary glands of ixodid ticks, it was unlikely that degeneration was complete by day 5. We now provide evidence that a decrease in secretory competence is due, in large part, to the loss of type III acini. Interestingly, only the granular acini exhibit apoptosis during the first two weeks off-host. This leads us to speculate on the function of the type I acini during the preoviposition period. We hypothesize that the type I acini are likely to be involved in maintaining the hydration state of the female during this critical period. Previous studies indicate that type I acini in females held to approximately the end of oviposition do not appear different from those of the unfed instars (Kahl et al., 1990). Together, the data suggest that DNA fragmentation begins in the granular acini after day 3 and continues throughout the post-detachment phase. We have no evidence thus far for mechanisms other than apoptosis in the acini, but are continuing this exploration. Further, it is not clear as to the fate of the epithelial cells surrounding the duct lumen, but we presume that based on the timing of their viability (at least through day 33) versus the end of oviposition, these tissues may die by necrosis. Acknowledgements This work was supported by a grant from the Professional Staff Congress of The City University of New York (63225). The work presented is a partial fulfillment of the requirements for an M.S. in Biology (L.J.). We wish to thank Dr. Lewis Coons of the University of Memphis for supplying the salivary glands in this study. References Ankarcrona, M., Dypbukt, J.M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S.A., et al., Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron 15, Coons, L.B., Lamoreaux, W.J., Developmental changes in the salivary glands of male and female Dermacentor variabilis (Say) during feeding. In: Borovsky, D., Spielman, A. 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