Modulation of the PGE2-Mediated Pathway in the Eclosion Blocking Effect of Flumethrin and Terpenoid Subfraction Isolated from Artemesia nilagirica in Rhipicephalus annulatus

The immunoperoxidase staining of the ovary of ticks treated with methanol (control), flumethrin and terpenoid subfraction using anti-COX1 antibodies are depicted in Figure 2A–C respectively. The ovarian tissues of the ticks treated with methanol, flumethrin and terpenoid subfraction did not reveal the presence of the COX1 enzyme.

The role of COX1 and COX2 enzymes in the reproduction was previously explained using the knockout mouse model. The mice deficient in COX1 exhibited longer gestation periods, protracted parturition and delivery of few live young mice compared to wild mice. The conception and fetal development were unaltered in the mice deficient with COX1 indicating that the prostanoids synthesized by COX1 were not important for ovulation, fertilization or implantation, but were essential for parturition. However, when there was ablation of the COX2 gene in mice, multiple reproductive failures, ovulation, fertilization, implantation and decidualization were noted, confirming that the prostaglandins synthesized by COX2 played an important role in these reproductive processes. Thus, both COX isoforms catalyzed the same reaction but then differed in their distribution in the reproductive tract [76].

The synthesis of the prostaglandins occurred through membrane-derived arachidonic acid (AA) via three sequential enzymatic reactions involving phospholipase A₂ enzymes, cyclooxygenases and specific terminal prostanoid synthases. The segregated utilization of COX1 and COX2 were observed in the prostaglandin biosynthesis pathway even though they were present together in the same cells. The constitutive COX1 enzyme was mainly seen in immediate prostaglandin synthesis, which occurred due to stimulation of Ca²⁺ mobilizers and was functionally coupled with cytosolic prostaglandin E synthase (cPGES). The inducible COX2 was mainly seen in delayed prostaglandin synthesis and was functionally coupled with microsomal prostaglandin E synthase-1 and 2 (mPGES-1 and mPGES-2). The COX1 was mainly localized in the endoplasmic reticulum and perinuclear membranes, whereas COX2 was located in the perinuclear envelope. The cPGES was located mainly in the cytosol and was expressed in a variety of cells and tissues as it was linked with constitutive COX1. The mPGES-1 located in the perinuclear membrane and functionally coupled with COX2 was mainly targeted by cancer drugs. The mPGES-2 localized in the Golgi apparatus lead to the maturation of cytosolic enzymes, was linked with both COX1 and COX2 [77]. The biosynthetic enzymes COX2 and mPGES were the two inducible enzymes that facilitated PGE₂ synthesis in synergy. The COX1 enzymes needed higher concentrations of arachidonic acid for their proper functioning than COX-2 [78].

In the present study, COX1 could not be localized in the oocytes of the ovaries of R. annulatus ticks treated with methanol (control), flumethrin (90 ppm) and terpenoid subfraction (40 per cent). The section of these ovaries mainly revealed the presence of stage III, IV and V oocytes. The stage III oocytes were large, with cytoplasm filled with small yolk droplets, whereas stage IV and V contained large and fused yolk droplets respectively. Therefore, due to the presence of more lipid droplets, the intensity of staining of COX1 was too low to be detected. There are reports that the expression of COX1 varied with the stage of the oocyte and was more expressed in early and late previtellogenic stage oocytes [55,79]. Furthermore, the studies on the expression of COX1 in the ovary of the Ostrich revealed that the intensity of immunostaining of COX1 decreased with the transition of oocytes to the vitellogenic follicular stage [79]. Even though there was a decrease in the content of yolk droplets and an increase in the presence of vacuoles in the oocytes in the ovary of the ticks treated with flumethrin, the localization of COX1 could not be appreciated in the oocytes. Hence, a proper conclusion on the role of COX1 on the reproduction of ticks could not be made in the present study.

The results of the immunoperoxidase staining with anti-PGES antibodies on the ovarian sections of ticks treated with methanol, flumethrin and terpenoid subfraction are depicted in Figure 3A–C, respectively. The ovarian tissues of the ticks treated with methanol showed localization of PGES around the cytoplasm of stage III oocytes. The ovary of the ticks treated with flumethrin and terpenoid subfraction showed no localization of the PGES enzyme. In the ovaries of the methanol-treated tick, the intensity of PGES was more around the cytoplasm near to nuclear region. The findings indicated that biosynthesis of prostaglandin occurred normally in the control group and the prostaglandins had an important role in the maturation of ovaries and oviposition. Moreover, the histological features of the ovary of the ticks treated with methanol (control) were comparable to that of a normal ovary [73]. Since the localization of the inducible COX2 was not studied, it was difficult to specify the type of PGES localized in the ovarian section. Therefore, it was not possible to conclude whether the PGES linked to COX2 was inhibited in the oocytes of the ovaries of R. annulatus ticks treated with flumethrin and terpenoid subfraction of A. nilagirica.

The working standards with ascending concentrations of PGE₂ at 5, 10, 25, 50, 100, 250, 500 and 1000 pg/mL in methanol were analyzed using LC-MS-MS. The respective peaks with the area were calculated. The calibration curve of PGE₂ concentration versus time was plotted and a linear regression was performed on the data set. The regression coefficient (R²) calculated was 0.999.

Prostaglandin E₂ was detected in methanol at the wavelength 196 nm with a retention time of three minutes. Recovery of PGE₂ from tick ovaries was done using different concentrations viz., 1 ppm and 5 ppm spiked samples in methanol and the recovery % was 83.61 and 98.03 respectively.

The ovarian samples from the ticks treated with methanol (control), flumethrin and terpenoid subfraction of A. nilagirica showed the detection of PGE₂ in all the samples analyzed. In ticks treated with methanol, the PGE₂ level detected was 24 pg/tick. The ovary of the ticks treated with flumethrin revealed the PGE₂ levels below the detection limit (BDL). The ovary of ticks treated with terpenoid subfraction detected a mean PGE₂ concentration of 4 pg/tick. The Table 3 depicts the mean concentration of PGE₂ between the groups of the treated ticks.

Prostaglandin E₂ is the most common prostanoid found in both mammalian and non-mammalian species with a variety of bioactivities and has various pathophysiological functions [80,81]. The role of PGE₂ in controlling the reproduction was well established in insects, fish, crustaceans, arthropods and mammals as well as in humans [53,54,55,56,82]. In ticks, PGE₂ is also involved in the regulation of ovarian maturation, oocyte development, egg-laying and associated behaviors in addition to the host-parasite relationship.

In the present study, the concentration of PGE₂ quantified in the ovaries of untreated R. annulatus tick was the 24 pg/tick. The concentration of PGE₂ levels in B. microplus ovaries was reported previously as 27 pg/tick [83]. The results indicated that there was not much variation in the levels of PGE₂ in both species of ticks. On the other hand, the concentration of PGE₂ was below the detectable limit in the ovaries of ticks treated with flumethrin. Moreover, the levels of PGE₂ were reduced to 4 pg/tick in the ovaries of ticks treated with the terpenoid subfraction of A. nilagirica.

In another study conducted in our laboratory, it was observed that flumethrin at concentrations ranging from 30–100 ppm caused a complete inhibition of fecundity in ticks [84]. In the present study, the inhibition of fecundity of ticks caused by treatment with terpenoid subfraction of A. nilagirica was 62%, which was comparatively less than the effect caused by flumethrin. These findings indicated that there was a significant difference in the inhibition of PGE₂ synthesis caused by the treatment with flumethrin and the terpenoid subfraction of A. nilagirica on ticks. Additionally, the few eggs laid by the ticks treated with the terpenoid subfraction could be correlated with the meagre quantity of PGE₂ detected in the treated tick ovary. Correspondingly, the key enzyme PGES involved in the biosynthesis of PGE₂ could not be detected in ovaries of the ticks treated with the flumethrin and terpenoid subfraction.

Many authors previously reported the gradual increase in the amount of PGE₂, PGF_2α and several unsaturated fatty acids during vitellogenesis in the ovary of Florida freshwater crayfish [51,85]. The concentration of PGE₂ in different stages of ovarian tissues of marine organisms varied from 20 ng/g tissue in the early stages to the highest levels in stage IV vitellogenic oocytes at 30 ng/tissue [86]. A very recent study demonstrated the implication of PGE₂ in inducing vitellogenesis, ecdysteroidogenesis and MF synthesis in selected edible crustaceans [56]. The results from the present study thus provide strong evidence for PGE₂ as an important molecule in signaling the oocyte maturation and in the laying of eggs in ticks. The data further support the concept that the reduced PGE₂ levels in the ovaries of ticks treated with flumethrin and terpenoid subfraction could be one of the reasons for the complete/partial eclosion blocking effect elicited. Thus, like other non-insect arthropods, PGE₂ is highly critical for reproduction in ticks.

The powdered plant material (2 kg) was extracted using ethanol for eight to nine refluxes in Soxhlet’s apparatus and was reduced and concentrated under the pressure of 175 mbar at a temperature range of 65 °C in a rotary vacuum evaporator (M/s Buchi, Flawil, Switzerland). The ethanolic extract thus obtained was air-dried and was fractionated in a separatory funnel using hexane (polarity index: 0.1) to yield hexane fraction. The dried hexane fraction was subjected to isolation of terpenoids using the Herz-Hogenauer method [90]. Approximately 20 g of the dried hexane fraction was soaked overnight in dichloromethane (100 mL) and sonicated. The slurry product was then filtered and the green filtrate was evaporated to dryness in a rotary vacuum evaporator (M/s Buchi, Flawil, Switzerland). Furthermore, the residue obtained was dissolved in 95% ethanol (70 mL) and warmed at 70–80 °C to increase the solubility in a thermostatic water bath (M/s Thermo Scientific, Waltham, MA, USA). The aqueous solution (5%) of lead acetate was added to this in a dropwise manner (10 mL) for the precipitation of fatty acids, phenolics and chlorophyll. The precipitate was removed by filtration using a pad of silica gel (230–400 mesh, M/s Merck India Ltd., Mumbai, India). The filtrate was kept in the thermostatic water bath (M/s Thermo Scientific, Waltham, MA, USA) at 40–50 °C until the viscous mass remained. The subfraction obtained was stored at 4 °C and used for further chromatographic or spectral analysis and biological assays.

The terpenoid subfraction isolated from the hexane fraction of the ethanolic extract of A. nilagirica was analyzed by a single quadruple GCMS-QP-2010 Ultra, (Shimadzu Corporation, Kyoto, Japan) with an RXi 5 SILMS (Restek Corporation, Bellefonte, PA, USA) 30 m × 0.25 mm column having an internal diameter of 0.25 µm. Different concentrations of the terpenoid subfraction (10 ppm, 25 ppm, 50 ppm and 100 ppm) were prepared in one milliliter of hexane and from these working solutions, one microliter was injected into the GC-MS system in the split mode (split ratio 1:10). Helium was used as the carrier gas with a flow rate of 1 mL/min. The column oven temperature was maintained at 110 °C for two minutes. Then, it was programmed to 220 °C at a ramp rate of 10 °C/min. The final temperature of 280 °C was attained at a ramp rate of 5 °C/min and it was then held for nine minutes. The injector and detector temperatures were optimized at 250 and 280 °C, respectively. The MS operating parameters were as follows: ionization energy, 70 eV; ion source temperature, 280 °C; solvent delay, 1.0 min; and scan range, 100 to 350 m/z. The total run time was 36 min. The components were identified based on matching their retention indices available with the mass spectral library (Wiley 08, NIST 11).

The adult immersion test was performed based on Drummond et al. [] The different concentrations of terpenoid subfraction (10% to 50%) were prepared in methanol. Four replicates, each with six ticks, were used for each concentration. The groups of six ticks selected randomly based on the size were weighed before the experiments and were immersed for two minutes in the respective dilution in a 50 mL beaker containing 10 mL extract. The ticks were recovered from the solution, dried using absorbent paper and placed in a separate plastic specimen tube (25 mm × 50 mm). The tubes were incubated at 28 ± 2 °C temperature and 80% relative humidity in a Biological Oxygen Demand (BOD) incubator. The adult tick mortality was observed up to the 15th day post-treatment. After oviposition, the eggs laid by the female ticks were collected and weighed. The eggs were kept under the same incubation conditions in a BOD incubator for the next 30 days. ⁹¹

The index of egg-laying (IF) and per cent inhibition of fecundity (InF) were calculated [] as follows.Percent inhibition of hatching of eggs. ⁹² (1) Index of egg laying ( ) IF = Weight of eggs laid ( ) g Weight of females ( ) g (2) Percentage inhibition of fecundity ( ) InF = [ ] IE ( ) control group IE ( ) treated group − × 100 IE ( ) control group

The eggs laid by ticks of each tube were weighed and observed for the next 24 days in the BOD incubator for visual hatching of larvae.

The dose-response data were analyzed by the probit method [93] using the Graph Pad Prism 4 software (Graph Pad Software Inc., San Diego, CA, USA). The LC₅₀ of the terpenoid subfraction was calculated by applying regression equation analysis to the probit-transformed data on the adult tick mortality and log concentration. The calculated LC₅₀ value of terpenoid subfraction was used for assessing the modulation of the prostaglandin-mediated pathway. The synthetic pyrethroid flumethrin was used as a positive control. Flumethrin at 90 ppm was reported with 100 per cent inhibition of fecundity and nil hatching percentage [84].

Thirty fully engorged adult female ticks were used for studying the localization of COX1 and PGES enzymes in the tick ovary by immunoperoxidase assay. They were divided into three groups of ten ticks each and immersed for two minutes in a separate beaker containing 10 mL methanol 100% (Group I), flumethrin, 90 ppm (Group II), and terpenoid subfraction, 40% concentration (Group III) respectively. The ticks were then recovered from the respective solutions, dried on absorbent tissue paper towels and placed in separate plastic specimen tube (25 × 50 mm). They were kept at 28 ± 1 °C and 85 ± 5% relative humidity (RH) in a BOD incubator (M/s Kemi, Ernakulam, India). After 24 h, they were taken out from the incubator, dissected in 0.9% saline solution and the ovaries were removed under a stereo zoom microscope (M/s Labomed, Gurgaon, India).

The processing of the ovarian tissues of the tick was performed as per the method described by Sumpownon et al. [55] with minor modifications. The dissected tick ovarian tissues were collected and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS 0.1 M) and kept at 4 °C overnight. The tissues were processed in ascending concentrations of ethanol. Paraffin-embedded blocks were made and sections were taken at 3 µm thickness with low-profile microtome blades. These sections were deparaffinised by xylene. The descending concentrations of ethanol were used for the rehydration of tissues. The sections were then incubated in the dark in 3% H₂O₂ containing methanol at room temperature for 15 min to suppress the endogenous peroxidase. The sections were then washed with PBS and incubated in PBS containing 0.1% glycine for 15 min and washed again with PBS. The slides were incubated in blocking solution (PBS containing 4% bovine serum albumin, 5% normal goat serum and 0.4% Triton X-100) to arrest the nonspecific bindings for one hour at room temperature. The tissue sections were washed thrice with PBS. Then the sections were incubated overnight in rabbit polyclonal anti-COX1 and/or anti-PGES antibodies diluted at 1:200 in PBS and Tween 20 (PBST) separately. These sections were again incubated with Horseradish peroxidase (HRP) secondary antibody i.e, HRP-conjugated goat anti-rabbit IgG at 1:500 in PBST for two hours. The staining of the sections was developed using DAB substrate solution for 15 min. Some of these sections were counter-stained with hematoxylin and eosin for identification. Ultimately, the immunoperoxidase stained sections were examined under an inverted fluorescence microscope (Model: Axio Vert.A1 FL-LED, M/s Carl Zeiss Microscopy, Oberkochen, Germany) for the localization of enzymes, COX1 and PGES. The images were captured and analyzed using ProgRes^® CapturePro-Camera Control Software. (JENOPTIK/Optical Systems Version 2.8.8., Goeschwitzer Strasse, Germany).

The iMark^TM microplate absorbance reader (M/s Bio-Rad, Hercules, CA, USA) with an absorbance range of 400–750 nm and the Microplate Manager^® 6 Software were used for the analysis. Thirty engorged female ticks were used for the quantification of vitellogenin (Vg) in the hemolymph of the tick by indirect enzyme-linked immunosorbent assay (ELISA). They were divided into three groups of ten ticks each and immersed for two minutes in separate beakers containing 10 mL methanol (Group I), flumethrin, 90 ppm (Group II), and terpenoid subfraction, 40% (Group III) respectively. Ticks were then incubated in BOD. After 24 h, the hemolymphs of ticks of all groups were collected using 10 µL microtips individually into 0.2 mL microtubes from the tick’s legs cut at the distal joint with the scissors under a stereo zoom microscope. The collected hemolymph was then diluted with 10 µL 1X PBS buffer. The concentration of protein in the hemolymph was measured in a spectrophotometer (M/s Thermo Scientific™ NanoDrop 2000, Waltham, MA, USA) at 280 nm absorbance. The concentration was adjusted to 2 µg/mL using a coating buffer (30 mM sodium carbonate and 75 mM sodium bicarbonate, pH 9.6). Fifty-six wells of ELISA plates were coated with the coating buffer containing hemolymph (100 µL) and incubated at 37 °C for 1 h or kept overnight at 4 °C. The plates were washed thrice with 1X PBS-Tween 20 (0.05%). Then, the plates were blocked with 1X PBS containing 3% skim milk powder (300 µL) and incubated at 4 °C overnight. After overnight incubation, the plates were again washed thrice with 1X PBS-Tween 20. Anti-vitellogenin antibody (diluted 1:300 in 1X PBS containing 1% skim milk powder) was added to each well (100 µL) and incubated at 37 °C for one hour. The plates were again washed 5 times with PBS-Tween 20. The Rabbit anti-mouse IgG-HRP conjugate (diluted 1:3000 in 1X PBS containing 1% skim milk powder) was added to each well (100 µL). The plates were then incubated at 37 °C for one hour and again washed five times with PBS-Tween 20. Orthophenylenediamine (OPD) was dissolved 1 mg/mL concentration in the substrate buffer (0.1 M citric acid and 0.2 M disodium hydrogen phosphate). The freshly prepared substrate solution was taken in a trough and H₂O₂ (3%) was added. The final substrate solution (100 µL) was added to each well and incubated for 10 min in the dark. After incubation, 50µL of 3N HCl was added to each well to stop the reaction. The absorbance was read at 450 nm in an ELISA plate reader (M/s Bio-Rad, Hercules, CA, USA).