Secretory Behavior of Salivary Glands of Rhipicephalus sanguineus Fed on Immunized Rabbit Hosts

The histochemical analysis of the salivary glands of Rhipicephalus sanguineus females (Latreille, 1806) (Acari, Ixodidae) fed for 2, 4 and 6 days (test group= TG) on hosts (New Zealand White rabbits) previously immunized with glandular extract of salivary glands obtained from females fed for 6 days (SGE6) was carried out in order to detect alterations in the secretion of glycoproteins, lipoproteins, acid phosphatase and calcium. It was here verified that the composition of the secretion produced by the glands of females from TG (2-6 days) was different from the one observed in those from control group (CG), with a gradual reduction in the synthesis of the components as feeding progressed, except for the proteins and calcium, which remained unaltered within the interval of 4 to 6 days and 2 to 4 days respectively. In the glands of females fed for 2 days (TG) there was increase in calcium staining, as well as a reduction in lipids and acid phosphatase, and staining for proteins and polysaccharides remained unaltered. In the females fed for 4 days (TG) there was a reduction in the intensity of staining of components, presenting an increase only for polysaccharides and calcium remained unaltered. In the glands of females fed for 6 days (TG) there was a reduction in the synthesis of the components, with an increase in proteins and calcium. Contrary to expected, it was verified that the alterations were more significant in the glands of females fed for 2 days than in the ones fed for 6 days, once the hosts were inoculated with antigens of the glandular tissue obtained from females fed for 6 days. In addition, it was verified that during the secretory cycle of glands from TG (2-6 days) a, c2, c5, d and f cells were the ones which presented the most histochemical alterations.


Introduction
The ticks are extremely successful obligatory hematophage organisms, due to the adaptation of their salivary glands to the production of saliva with a high capability to modulate the immuneinflammatory and hemostatic systems of the infested host [1][2][3][4][5][6][7][8].
The meaning of hematophagy for the hosts goes beyond the spoliative action of the ectoparasite, being also related with the vehiculation of pathogenic agents due to the immunosuppressing properties of the ticks' saliva [9][10][11].
In the tick-host relationship it is verified that the saliva of the ectoparasite undergoes alterations in its composition [12][13][14], consequence of the alterations in the secretory behavior of its own secretory glands, which occurs in order to modulate the host according to the ectoparasite's needs [15].
In the case of R. sanguineus females, whose blood feeding occurs in the period of 7-8 days, their salivary glands undergo alterations throughout the process. Such glands comprise acini I, II and III, and the latter two undergo the most alterations, once they act in the feeding and osmoregulation processes in the phase of high blood consumption [16]. In the salivary glands of fasting females a, c1, c3, d and e cells are present. In those fed for 2 days b, c2, c4, c5, c6 and f cells are activated; in those fed for 4 days c5 and c6 cells start to lose activity and f cells became inactive from the secretory point of view; in the individuals fed for 6 days the glands are already in degeneration; and b, c5 and c6 cells are completely inactive [17].
However, it is known that the feeding by R. sanguineus female ticks on previously immunized hosts alters the secretory behavior according to the composition of the extract used for the immunization of the hosts. In this sense, an inactivity of some cellular types is verified, as well as precocious degeneration of the gland as a whole, events which occur to compensate the resistance acquired by the immunized hosts and allow the ticks' complete blood feeding [16].
Thus, the present study had the objective to histochemically follow the secretory cycle of R. sanguineus females fed for 2, 4 and 6 days on rabbits previously immunized with extracts of salivary glands from females of the same species fed for 6 days (SGE6).
Thus, the present study aimed to evaluate the histochemical changes in the secretory cycle of R. sanguineus females fed for 2, 4 and 6 days on rabbits previously immunized with extracts of salivary glands obtained from females of the same species fed for 6 days (SGE6) using techniques for detection of proteins, polysaccharides , lipids, acid phosphatase and calcium.

Materials
To perform this study salivary glands of adult R. sanguineus females fed for 2, 4 and 6 days subjected to infestation on New Zealand White rabbits immunized with glandular extract of females fed for 6 days were used. For this, unfed individuals (males and females) from a colony kept in BOD incubator, in controlled conditions (29°C, 80% of humidity and photoperiod of 12 hours), in the Biotery of the Biology Department of UNESP campus Rio Claro (SP) were used in the infestations A, B e C made in rabbits according to the procedure described in the literature [18].
Infestation A: made in naive rabbits using 25 couples of R. sanguineus couples/host, for the acquisition of females of ticks fed for 6 days (25 individuals), for the obtainance of glandular extract: SGE6= glandular extract of females fed for 6 days.
The extract was processed and inoculated in the hosts subjected to infestation B.

Methods
Obtaining Salivary Glands and EGS6 Glandular Extract: In the Laboratory of the Department of Molecular Biology of UNESP Rio Claro (SP), Brazil, the salivary glands were put in eppendorf tube containing 200 µL of phosphate buffer pH 7.4. Then the glands were macerated, centrifuged for 30 minutes at 10.000 xg, the supernatant was collected and was put for proteins dosage, which occurred according to the methodology described in the literature [19] (Bradford method), which should be of at least 0.2 µg/µL.
After the determination of the protein content, the extract was filtered with the help of sterile filtering units (JBR610303, disposable filtering unit Millex GV, durapore membrane PVDF, Millipore, MilliUni), of 0.22 µm and 13 mm of diameter, attached to hypodermic syringes in the interior of a pre-sterile vertical laminar flow. The extract was then divided in volumes of 50 µL and kept in freezer at -20 o C. Only at the moment of inoculation the extract was mixed (50µL of extract/ host) with 50 µL of complete Freund adjuvant (reference # F 5881, Sigma-Aldrich), procedures that are also made in pre-sterile vertical laminar flow.

Immunization of Rabbits Hosts and Realization of the Challenge Infestation:
After, rabbits from TG had the right dorsal side sheared and were subcutaneously inoculated with SGE6 extract, via hypodermic syringe, for three times in intervals of 21 days. Only after 15 days from the last inoculation, all the hosts from TG and CG were subjected to infestation challenge with 15 couples of R. sanguineus/host.
In the sequence, R. sanguineus females fed for 2, 4 and 6 days were removed from the rabbits inoculated with SGE6 extract (GT), as well as from those not inoculated (CG) and subjected to histochemistry analysis.

Histochemical Analyses:
In the Histology Laboratory of the Biology Department of UNESP campus Rio Claro (SP), Brazil, salivary glands of R. sanguineus females of each group (TG and CG) fed for 2, 4 and 6 days were removed in buffered saline solution (7.5 g NaCl + 2.38 g Na 2 HPO 4 + 2.72 g KH 2 PO 4 in 1000 mL distilled water) and fixed.
Part of the material was fixed in 4% paraformaldehyde at 4 o C, after fixation, the material was dehydrated in a series of increasing concentrations of ethanol (70%, 80%, 90% and 95%), embedded in resin (Leica), and sectioned at 3 µm thickness. Sections were mounted on glass slides and histochemical tests were applied to detect the presence of the following compounds: proteins (bromophenol blue) [20]; polysaccharides (PAS-Periodic Acid Schiff) [21]; calcium (von Kossa) [22] and lipids [23].
The other part of the material was fixed in 10% buffered neutral formalin and acetone (9:1) for one hour and thirty minutes at 4 o C, then processed according to the methods described by literature [24] for detection of acid phosphatase activity. The material was then dehydrated in increasing concentrations of ethanol (70%, 80%, 90% and 95%), embedded in resin (Leica), and sectioned at a thickness of 7µm. The sections were placed on glass slides and counterstained with hematoxylin for 2 minutes. The control samples were incubated without substrate.
The slides of the all histochemical tests were mounted with Canada balsam and examined and photographed under Motic BA 300 light microscope.

Results
Acini I were not considered in the analysis of salivary glands of the control group (CG) and test (TG), once they are agranular and do not have a secretory function, acting exclusively on the hydric balance of the ectoparasite [25][26].

Control Group (CG)
The histochemical data for the salivary glands of R.sanguineus females fed for 2, 4 and 6 days from the control group (CG) were presented in study realized previously [27].  Acinus III:

Females fed for 6 days
Acinus II: -a cells: the granules are moderately stained for bromophenol blue ( Figure 1M and 1N) and Baker ( Figure 3P) and negative for PAS ( Figure 2M-O), acid phosphatase ( Figure 4L, 4N and 4O) and von Kossa ( Figure 5N and P).
-c4 cells: it was not possible to observe these cells in this feeding phase through the techniques applied.
-c5 cells: are not observed for being inactive in this phase.
The results for salivary glands of the females from groups CG and TG fed for 2, 4 and 6 days are summarized in Tables 1-8.

Discussion
In this study, a histochemical analysis of the salivary glands of R. sanguineus females fed for 2, 4 and 6 days in hosts previously immunized with the glandular extract obtained from salivary glands of females of the same species fed for 6 days (SGE6) was performed, with the objective to detect alterations in the secretory cycle of glycoproteins, lipoproteins, phosphatase and calcium, commonly present in the saliva of these individuals.
Considering the results obtained it was verified that the ticks' feeding on host immunized with SGE6 extract affected the secretory behavior of the gland, corroborating [28], who verified that the pharmacology of the ticks' saliva could be molded according to the specific haemostatic defenses of the host, as the composition of the saliva would undergo variations in the composition throughout the glandular cycle [12-14, 17, 29-31], satisfying the ectoparasite's needs in the modulation of the immune-inflammatory and hemostatic systems of the host. The data here The results for the salivary glands females fed for 2, 4 and 6 days from the CG were presented in the study by  obtained demonstrated that the hosts of TG group developed resistance to the glandular antigens present in SGE6 extract used in the inoculation.
In this study a comparison of histochemical data from different periods of the secretory cycle (2, 4 and 6 days) of glands from TG group The results for the salivary glands females fed for 2, 4 and 6 days from the CG were presented in the study by The results for the salivary glands females fed for 2, 4 and 6 days from the CG were presented in the study by  was made and alterations between one period and another in relation to the same comparison made in CG group were verified. In this sense, in CG group a gradual reduction in the components as the feeding process progressed was observed, except for calcium and proteins which remained unaltered in 4-day glands, in relation to the 2-day glands; and in the 6-day Study Groups CG TG 2 days of feeding 4 days of feeding 6 days of feeding 2 days of feeding 4 days of feeding 6 days of feeding Acini Cells The results for the salivary glands females fed for 2, 4 and 6 days from the CG were presented in the study by

Study Groups
CG TG 2 days of feeding 4 days of feeding 6 days of feeding 2 days of feeding 4 days of feeding 6 days of feeding Acini Cells The results for the salivary glands females fed for 2, 4 and 6 days from the CG were presented in the study by Furquim et al. (submitted paper). *: inactive cell; φ: not observed cell; __: no secretion granules; the intensity of staining varied from -, being: : negative and : strongly positive. Study Groups CG TG 2 days of feeding 4 days of feeding 6 days of feeding 2 days of feeding 4 days of feeding 6 days of feeding Acini Cells The results for the salivary glands females fed for 2, 4 and 6 days from the CG were presented in the study by Furquim et al. (submitted paper).*: inactive cell; φ: cell not observed; __: no secretion granules; the intensity of staining varied from -, being: : negative and : strongly positive. glands in relation to the 4-day ones, respectively. In TG group the glands of females fed for 4 days, compared with the ones fed for 2 days, presented a reduction in proteins, acid phosphatase and calcium; and the glands of females fed for 6 days, compared with ones fed for 4 days, showed a reduction in polysaccharides, lipids and acid phosphatase. This glandular behavior could be explained by the parasite-immunized host relationship; i.e., the ectoparasites responded modifying the composition of their saliva according to the immunologic response of the host, which in this case developed resistance against the antigens present in SGE6 extract.
As for the histochemical alterations detected in each specific period of the secretory cycle (2, 4 or 6 days) in TG group, it was observed that in the glands of females fed for 2 days there was an increase in calcium, reduction of lipids and acid phosphatase and the intensity of staining for proteins and polysaccharides remained unaltered. In those fed for 4 days there was mainly a reduction in the staining of components, except for polysaccharides, which increased; and calcium, which remained unaltered. In those fed for 6 days a reduction in the components was also detected, except for proteins and calcium, which increased. This showed that the immunization with SGE6 glandular extract caused histochemical alterations in the glandular secretion, which would be mainly related to the reduction of the component contents here analyzed.
As for the cellular types, in a decrescent order of alterations, the most affected cells were: as c5; a, f; d; c2; and c3, e in the females fed for 2 days; d; c5; a, c1, c2, e in the females fed for 4 days; and c2; a, c1, d; e in the females fed for 6 days. According to literature a and d cells would be responsible for the synthesis of lipoproteins which form the cement [29], in addition to proteins, from which some would be antigenic [32], and would act both in modulation of the host and the formation and maintenance of the feeding lesion [30]. The secretions synthetized by c and f cells would be involved in the modulation of the local hemostatic reactions of the host [29,[33][34] and the glycoproteins produced by c cells would act as anticoagulants [34].
In this sense, concerning the secretions of the most affected cells it can be inferred that those synthetized by a, c2 and d cells would present high immunologic capability when introduced in the host through inoculation of SGE6 extract, once this extract presented different biochemical and pharmacological characteristics from any other extract from other periods of the secretory cycle. This has probably occurred due to the quantitative and qualitative variation of its molecules [27]. However, for the secretory behavior of c5 and f cells, a possible explanation would rely on the importance of their secretion to the initial modulation of the immune-inflammatory and hemostatic systems of the host, and not to its immunogenicity, a hypothesis based on the fact that the hosts considered have not been previously immunized with the antigens from c5 and f cells, as they were not active in the glands of females fed for 6 days (glandular tissue used in the production of SGE6 extract), demonstrating that the hosts could not produce antibodies against these antigens.
In addition to this, it was verified that the components of the glandular secretion which underwent the most alterations were the lipids, followed by proteins, polysaccharides/acid phosphatase and calcium. According to literature, the lipid elements present in the ticks' saliva would be in the form of prostaglandins [4,13,[35][36], purine nucleoside adenosine [36] and prostaciclins [37], molecules which would be part of the sophisticated pharmacologic arsenal for the modulation of the local hemostatic reactions in the host, which actuation would affect the ectoparasite's capability to remains attached and consuming blood [8]. This could explain why the lipid staining was the one which presented the most modifications in this study.
The data presented above confirm that the most pronounced alterations occurred in the glands of females fed for 2 days, and the alterations in 4-day and 6-day glands were less intense. The information provided here is quite inusitate, once it was expected that the most affected glandular tissue would be the one obtained from females fed for 6 days, once antigens from this tissue were inoculated on the hosts ( SGE6 extract). Such information confirms the hypothesis that the hosts would respond differently due to the qualitative and quantitative variation of the antigens inoculated in them [27] and, in this sense, the immunization with SGE6 extract stimulated in the hosts the development of an immune response mainly against the salivary antigens of females fed for 2 days, where the most pronounced histochemical alterations occurred.