MATERIALS AND METHODS

Tick preparation: A total of 90 R. sanguineus semi-engorged females, average weight 30 mg, from one dog were used in this experiment. The ticks were separated into groups of 10, with 3 repetitions for each treatment with (1) The acaricide fipronil (0.25% w/v), (2) Cassia fistula ripe pod extract and (3) Control groups. Before treatment, all ticks were morphologically examined under a stereoscope (Nikon, Tokyo, Japan). The experiment was reviewed and approved by the Institutional Animal Care and Use Committee, Mahasarakham University (approval number: 0026/2017).

Plant extract preparation: The ripe pods of C. fistula were collected from Maha sarakham province, Thailand in April, 2017. A decoction extract was processed following the method of Sakulpanich and Gritsanapan²⁴. About 10 g of the fresh ripe pod was separated from C. fistula pods and boiled with 100 mL distilled water for 1 h at 95-98°C. The mixture was filtered through a Whatman size 1 filter paper and the pod was re-extracted several times until the extraction was paled. The decoction extracts were mixed, filtered again and the filtrate was evaporated to dryness on a boiling water bath to yield a dry plant extract. The dry crude extract was dissolved in distilled water at a concentration of 100 mg mL^–1.

Immersion test: Rhipicephalus sanguineus semi-engorged female ticks were washed with distilled water and then dried on absorbent paper. Afterwards, the ticks were immersed in test solutions in petri dishes for 5 min following the method given by Juasook et al.¹⁴. The three treatment groups consisted of 0.25% w/v fipronil (FP), C. fistula ripe pods extract (CE), while ticks in the control group (CON) were immersed in distilled water for the same time period. The ticks were dried on absorbent paper and placed in an incubator (Memmert, Schwabach, Germany) under controlled conditions, 28±1°C, RH >80% and a 12 h photoperiod, for 14 days.

Mortality and laying rate: The acaricidal effect on semi-engorged ticks was investigated and recorded everyday of the experiment for calculation of mortality rate and laying rate. Dead ticks were identified by loss of motility and pedal reflex. Dead ticks were fixed with Karnovsky's solution for scanning electron microscopic examination. The calculations used in this experiment were:

Scanning electron microscopy: To observe the surface structure of the tick post-treatment, ten dead ticks from each group were washed several times with 0.2 M cacodylate buffer (pH 7.2) and fixed with Karnovsky’s solution at 4°C overnight. After washing for 10 min, 3 times, with the phosphate buffer, samples were post-fixed with 1% OsO₄ in 0.1 M phosphate buffer (pH 7.2) for 1 h, followed by washing again for 10 min, 3 times, in phosphate buffer. Samples were then dehydrated through a graded ethanol series (30, 50, 70, 90, 95 and 100% alcohol, 2 times), dried with a critical point dryer, coated with gold using a JFC-1100E ion sputtering device (JEOL, Tokyo, Japan) and observed with a JEOL JSM-7800F scanning electron microscope (SEM) at a 1.0 kV accelerating voltage.

Statistical analysis: The mortality rate and laying rate were analyzed by one-way ANOVA (SPSS version 22.0). Tick survival was also evaluated by Kaplan-Meier analysis. A p<0.05 was considered statistically significant.

RESULTS

Laying rate: The effect of each treatment on the oviposition of R. sanguineus semi-engorged females was examined by the laying rate, which was observed during incubation of ticks. This experiment showed that fipronil had the highest acaricidal activity, completely inhibiting oviposition of female ticks. Interestingly, the C. fistula ripe pod extract had efficacy in controlling egg laying by ticks, although with lower activity than the chemical product. The laying rates of female ticks treated with extracts of the C. fistula ripe pod extract were 33.33%, significantly less (p<0.05) than the control group (50.00%) (Fig. 1).

Mortality rate: Ticks received various treatments and were then incubated to study the mortality rate. After treatment, dead ticks were identified by loss of motility and pedal reflex and the number of deadticks in each group was observed and recorded everyday post-treatment (Fig. 2). The tick mortality in all groups started at days 4-6 post-treatment with peak mortality on days 9-10. Remarkably, this study revealed no significant difference of mortality rate in the FP and CE groups (100 and 83.33%, respectively). At the end of the experiment, the lowest mortality was found in the CON group (46.67%).

Fig. 1:	Laying rates of R. sanguineus semi-engorged females
	FP: Treated with fipronil, CE: Treated with C. fistula pod extract, CON: Control, a-c indicated a significant difference at p<0.05

Fig. 2:	Mortality rates of R. sanguineus semi-engorged females
	FP: Treated with fipronil, CE: Treated with C. fistula pod extract, CON: Control, a-b indicate a significant difference at p<0.05

Moreover, Kaplan-Meier analysis of survival of R. sanguineus semi-engorged females (Fig. 3) showed median survival time in the FP group and the CE group (9 and 10 days, respectively). Median survival time in the control group was longer than the both of the other groups which can not be evaluated from the short duration of the experiment.

Scanning electron microscopy: In the present study, dead female ticks in all experiments were fixed and processed for analysis of acaricidal activity on the morphological changes of R. sanguineus semi-engorged females. All ticks in this study showed normal external structure, except that ticks exposed to fipronil as a chemical acaricide showed a thin and smooth surface which ticks in the natural treated group and control group had a thick and wrinkled surface (Fig. 4).

Fig. 3:	Kaplan-Meier analysis of surviving R. sanguineus semi-engorged females in each treatment group during the experiment
	FP: Treated with fipronil, CE: Treated with C. fistula pod extract, CON: Control, There were significances in overall survival between all treated groups and control group (p<0.05), Hash marks indicate censored events

Fig. 4(a-c):	Scanning electron micrographs of R. sanguineus semi-engorged females ventral view, (a) Treated with fipronil, (b) Treated with C. fistula pod extract and (c) Control
	Cp: Capitulum, A: Anus

DISCUSSION

Considering that R. sanguineus tick has become an important ectoparasite, it is desirable to investigate new methods of control aiming to find alternatives which are less aggressive to the environment and non-target organisms. This study investigated the acaricidal activity of a natural plant, C. fistula ripe pod extract, on laying, mortality and morphology of R. sanguineus semi-engorged females compared with fipronil.

Fipronil, is popular for use in tick management in dogs and is a widely accepted acaricide belonging to the phenylpyrazole family²⁵. It interferes with the central nervous system of insects by acting mainly in the GABA system^10,26,27. In this study, ticks treated with fipronil had absolutely mortality and oviposition was entirely inhibited for 14 days after treatment, in agreement with a previous report by Juasook et al.¹⁴ and De Oliveira et al.²⁸. The Scanning Electron microscopy showed a thin and smooth surface of a semi-engorged tick. The mechanism of action of fipronil is not restricted to the tick’s nervous system but also inhibits the feeding process by damaging the salivary gland¹². Therefore, the actions of fipronil on tick’s surface are needed to further studies. This result supported previous studies showing that fipronil also has an impact on the reproduction of the tick^28,29. Nevertheless, the toxicity of fipronil can contaminate the environment and damage non-target organisms, including animals and humans^10,30 and also has been classified as a possible human carcinogen^31,32. Therefore, many plant extracts were tested for their acaricidal effects.

Cassia fistula Linn. is a flowering plant which is widespread in Thailand and is known as Khun in Thai language. The whole plant possesses medicinal properties³³ and also antiparasitic, larvicidal and ovicidal activities^17,22. In Thai traditional medicine, the ripe pods have been used as laxative drug³⁴. The ripe pod contains several anthraquinones such as rhein, aloin, emodin, sennosides and aloe-emodin, both in aglycone and glycosides forms³⁵. The present study collected C. fistula ripe pods and decoction extracted them to investigate their acaricidal activity.

Even though, the acaricidal activity against female brown dog ticks by inhibiting laying rate is less than for the synthetic acaricide, C. fistula ripe pods extracts at 100 mg mL^–1 mortality was not significantly different from the fipronil group. From scanning electron microscopy, ticks in both the C. fistula ripe pods extracts and control group demonstrated similar morphology. To evaluated the mechanism of this plant extract for female tick controlling, ultrastructure microscopy need to further experiment.

Previous research, the ripe pod extract of C. fistula was also identified rhein as the major anthraquinone component as the leaf extract³⁴. Rhein, an anthraquinone derivative is known to exhibit a variety of biological activities such as antimicrobial, antifungal, purgative, anti-inflammatory, analgesic, anti-tumor and hypoglycaemic activities^34,35. Duraipandiyan et al.³⁶ demonstrated that rhein isolated from the ethyl acetate extract of C. fistula flowers exhibited antifeedant and larvicidal activities against cotton bollworm and leaf worm. In addition, the acaricidal effect of C. fistula leaf extract against Aedes aegypti, Culex quinquefasciatus, Anopheles stephensi and Rhipicephalus (Boophilus) annulatus were also reported by Sunil et al.¹⁵, Govindarajan¹⁷ and Govindarajan et al.²³. The acaricidal activity shown in this experiment could be attributed to the presence of the anthraquinone compound rhein.

CONCLUSION

The results of this study demonstrate that C. fistula ripe pod extracts affected the mortality and inhibited oviposition of R. sanguineus semi-engorged females, indicating that these plant extracts can be active against brown dog tick and could be a promising natural acaricidal product. However, the acaricidal property of the C. fistula has not been previously reported. Further studies are needed to identify the active ingredients present in the plant ripe pods and explore its possible mechanism for the inhibiting laying rate and the mortality of ticks.

SIGNIFICANCE STATEMENT

This study discovered that the C. fistula ripe pod extracts affected the mortality and inhibited oviposition of R. sanguineus semi-engorged females. Thus, this plant extract, which is a natural compound, could be a promising natural acaricidal product to control R. sanguineus in dogs. Therefore, the active ingredients present in this plant and its possible mode of action for the control of tick population should be defined in further studies.

ACKNOWLEDGMENTS

The authors are thankful to scientists of the Pathology unit, Teaching Animal Hospital and the Parasitology Laboratory, Faculty of Veterinary Sciences, Mahasarakham University, Maha Sarakham, Thailand, for their assistance.