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Eclosion blocking effect of ethanolic extract of Leucas aspera (Lamiaceae) on Rhipicephalus (Boophilus) annulatus
Veterinary Parasitology 179 (2011) 287–290
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Eclosion blocking effect of ethanolic extract of Leucas aspera (Lamiaceae) on Rhipicephalus (Boophilus) annulatus Reghu Ravindran a,∗ , Sanis Juliet b , A.R. Sunil a , K.G. Ajith Kumar a , Suresh N. Nair b , K.K. Amithamol a , M. Shynu c , Ajay Kumar Singh Rawat d , Srikanta Ghosh e a b c d e
Department of Veterinary Parasitology, College of Veterinary and Animal Sciences, Pookot, Wayanad, Kerala 673 576, India Department of Veterinary Pharmacology and Toxicology, College of Veterinary and Animal Sciences, Pookot, Wayanad, Kerala 673 576, India Department of Veterinary Biochemistry, College of Veterinary and Animal Sciences, Pookot, Wayanad, Kerala 673 576, India Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute, Lucknow UP226 001, India Division of Parasitology, Indian Veterinary Research Institute, Izatnagar UP243122, India
a r t i c l e
i n f o
Article history: Received 16 August 2010 Received in revised form 23 February 2011 Accepted 28 February 2011 Keywords: Leucas aspera Acaricidal activity Eclosion Rhipicephalus (Boophilus) annulatus
a b s t r a c t The crude ethanolic extract of aerial parts of Leucas aspera was tested for its acaricidal properties against Rhipicephalus (Boophilus) annulatus. The per cent adult mortality, inhibition of fecundity and hatching of laid ova were studied at concentrations of 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mg/ml. Adult tick mortality was significant at the highest concentration tested. Inhibition of fecundity of treated groups differed significantly from control and was concentration dependent. L. aspera extract also produced complete failure of eclosion of eggs from the treated ticks even at lower dilutions of the extract. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Ticks and the diseases they transmit are widely distributed throughout the world, particularly in tropical and subtropical regions. It has been estimated that 80 per cent of the world’s cattle population is exposed to tick infestation (FAO, 1984). The inherent problems of chemical acaricides like the resistance (Nolon, 1990) and environmental residues resulted in only partial success of tick control. The searches for environmentally safe products have accelerated the research on botanical acaricides. The active ingredients from plants are known to possess insecticidal, growth inhibiting, anti-moulting and repellent activities (Ghosh et al., 2007). Rhipicephalus (Boophilus) annulatus is a one host tick under subgenus Boophilus within the genus Rhipicephalus
∗ Corresponding author. Tel.: +91 9447713422; fax: +91 4936 256390. E-mail address: [email protected] (R. Ravindran). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.02.021
and occurs in greatest abundance in tropical and subtropical regions of the world. This cattle tick not only affects production but also acts as the vector of many protozoal, viral and rickettsial diseases (Wall and Shearer, 1997; Roberts and Janovy, 2005). Even though Rhipicephalus (Boophilus) microplus is the major tick affecting cattle of northern parts of India, R. (B.) annulatus is the commonest species found in southern India (Jagannath et al., 1979; Koshy et al., 1982; Rajamohanan, 1982). Leucas aspera (Lamiaceae) is a small herbaceous, erect plant with a free blooming nature that grows as a weed on waste lands and roadsides all over India. It is pungently aromatic and commonly used as antipyretic herb in south India. In traditional medicine it is indicated for cold, cough, painful swellings and chronic skin eruptions (Chopra et al., 2002). The plant was previously evaluated pharmacologically for its anti-inflammatory, analgesic and protective effects against cobra venom poisoning (Reddy et al., 1993a,b). Recently, Maheswaran et al. (2008) reported the larvicidal activity of the hexane extract of L. aspera against Culex quinquefasciatus and Aedes aegypti.
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R. Ravindran et al. / Veterinary Parasitology 179 (2011) 287–290
Table 1 Effects of different dilutions of ethanolic extract of L. aspera against R. (B.) annulatus. Sl no.
Concentration (mg/ml)
Mean tick weight per replicate ± SEM
1 2 3 4 5 6 7 8
Control 100 50 25 12.5 6.25 3.13 1.56
1.0182 1.0657 1.0665 1.0619 1.0736 1.0736 1.0098 0.9971
± ± ± ± ± ± ± ±
0.0382a 0.021a 0.0419a 0.0231a 0.0365a 0.0365a 0.0588a 0.0119a
Mean % adult mortality within 15 days ± SEM 0 54.16 45.83 8.33 4.16 4.16 4.16 4.16
± ± ± ± ± ± ± ±
0a 7.978b 7.978b 4.809a 4.165a 4.165a 4.165a 4.165a
Mean eggs mass per replicate ± SEM 0.4381 0.1419 0.1868 0.2601 0.2920 0.3018 0.3098 0.3280
± ± ± ± ± ± ± ±
0.0301d 0.0286a 0.0193ab 0.0371bc 0.0151c 0.0103c 0.0362c 0.0351c
Index of fecundity ± SEM
0.04299 0.1317 0.1753 0.2451 0.2757 0.2831 0.3049 0.3301
± ± ± ± ± ± ± ±
0.024d 0.0268a 0.0181ab 0.0370bc 0.0173c 0.0196c 0.0269c 0.0388c
Percentage inhibition of fecundity (%)
Hatching % (visual)
0 69.36 59.22 42.99 35.87 34.15 29.08 23.21
100 0 0 0 0 0 0 0
n = 4. Values are mean ± SEM, means bearing different superscripts a–d (P < 0.05), indicate significant difference when compared with the control.
The present study evaluates the potential of an ethanolic extract of L. aspera, as an acaricide against R. (B.) annulatus. 2. Materials and methods 2.1. Preparation of plant extract The L. aspera (Locally known as ‘Thumba’) were collected from Vythiri taluk of Wayanad district, Kerala. The voucher specimen was deposited in the herbarium of the National Botanical Research Institute, Lucknow. The aerial parts of the plant were cleaned and dried in shade at room temperature for two weeks. Dried plant material (1 kg) was pulverized using a pulverizer. The powdered plant (100 g) was used for ethanolic extraction in a soxhlet apparatus attached with solvent recovery unit (Rotavac, Buchi, Switzerland). Crude extract was dried at room temperature, weighed and dissolved in methanol for making the following dilutions of 1.56 mg/ml, 3.13 mg/ml, 6.25 mg/ml, 12.5 mg/ml, 25 mg/ml, 50 mg/ml and 100 mg/ml that were used for the testing. 2.2. Collection of ticks The fully engorged adult female ticks were collected from infested animals. The ticks were washed in tap water and dried on an absorbent paper. They were used for the adult immersion test (AIT). 2.3. In vitro acaricidal effects A total of 192 adult engorged female ticks were used for the present study. They were divided into eight groups (seven treatments and one control) each comprising of 24 ticks as six ticks each in four replicates. Each group was used to estimate the acaricidal effects of respective concentration of plant extract. The weight of each replicate of treated ticks was determined and immersed them for 2 min in 10 ml of the respective concentration of ethanolic extract. The control group was immersed in methanol. Ticks were recovered from the respective solution. They were dried using filter paper. Ticks of each replicate were placed in a separate specimen tube. They were incubated at 28 ◦ C and 80 per cent relative humidity (RH) in a BOD incubator. These ticks were observed for oviposition and death
up to 15 days. The per cent adult tick mortality and the weight of the eggs laid by the treated ticks were recorded in comparison with the control. The eggs were incubated at the same condition and the percentage of hatched eggs was estimated visually. The index of egg laying and percentage inhibition of fecundity were calculated using the following formulae (1) and (2), respectively (FAO, 2004; Goncalves et al., 2007): Index of egg laying (IE) =
weight of eggs laid (mg) weight of females (mg)
(1)
Percentage inhibition of fecundity (IF) (%) =
[IE (control group) − IE (treated group)] × 100 IE (control group)
(2)
2.4. Statistical analyses All the data were expressed as the mean ± SEM. Groups were compared using one-way ANOVA for repeated measurements using SPSS software. Duncan’s test was used for post hoc analysis. A value of P < 0.05 was considered significant. 3. Results and discussion The results of adult immersion test using the ethanolic extract of L. aspera are shown in Table 1. The efficacy of extract against R. (B.) annulatus females was assessed by estimating the per cent adult mortality, inhibition of fecundity and hatching rate. The per cent adult tick mortality caused by the ethanolic extract of the L. aspera varied from 4.16 to 54.16 per cent, when tested at concentrations ranging from 1.56 to 100 mg/ml. A statistically significant effect on adult mortality could be observed at higher concentrations of 50 mg/ml and 100 mg/ml. L. aspera has been used as an insecticide in traditional medicine in India. The larvicidal activity of L. aspera against mosquitoes was previously reported (Muthukrishnan et al., 1997; Maheswaran et al., 2008). They found first and second instar larvae of C. quinquefasciatus as more sensitive compared to third and four instars. Mwangi and Rembold (1988), reported that leaves of L. aspera exhibited heavy mortality especially during moulting process or subsequent process of melanization and tanning. Mangathayaru
R. Ravindran et al. / Veterinary Parasitology 179 (2011) 287–290
et al. (2006) isolated a liquid alkaloid from L. aspera and identified it as nicotine and speculated that the insecticidal property of the herb could be due to the presence nicotine. The results of the present study further demonstrated that the ethanolic extract of L. aspera induced a significant concentration dependant decrease in egg mass production. The inhibition of fecundity ranged from 23 to 69 per cent on the treated ticks. In ticks, the moulting hormone (ecdysteroids) plays a role in the regulation of salivary gland function, production of pheromones and oogenesis and oviposition (Rees, 2004). In fully engorged adult female ticks, the level of ecdysteroids rises in the haemolymph which in turn causes salivary gland to degenerate and as a result the ticks lay more eggs. This hormone also triggers vitellogenesis (Sankhon et al., 1999) and inhibits reattachment to the host (Weiss and Kaufman, 2001). The neuronal involvement in the control of salivary gland degeneration in the ixodid tick, Amblyomma haebraeum has been proved (Harris and Kaufman, 1984). The neurotransmitter dopamine regulates the synthesis of ecdysteroids. Dopamine was identified in the salivary gland and salivary gland nerves of R. (B.) microplus and A. haebraeum (Binnington and Stone, 1977; Kaufman and Harris, 1983). Dopamine binds to the D1 like receptor on the salivary glands to effect fluid secretion and synthesis of ecdysteroids. The dopamine receptor is coupled to a G-protein activated adenylate cyclase and the activation produces an increase in cAMP. The increase in cAMP regulates many aspects of cellular functions by activation of protein kinases. The protein kinases by catalyzing the phosphorylation of serine and threonine residues activate the target enzymes that synthesize ecdysteroids. The dopamine also opens a voltage gated Ca2+ channel allowing influx of extracellular Ca2+ to stimulate cytosolic phospholipase A2 to liberate arachidonic acid. This arachidonic acid is then converted by a cyclooxygenase pathway to Prostaglandin E2 (PGE2 ) and Prostaglandin F2␣ (PGF2␣ ) and small amounts of Prostaglandin I2 (PGI2 ) (Aljamali et al., 2003). PGE2 and PGF2␣ are found in very high concentration in tick saliva and salivary glands (Bowman et al., 1996). In addition, PGE2 plays a very important role in peristaltic activity of oviduct to transport the oocytes to the genital aperture. It interacts with the Eicosapentenoic acid (EP1 ) like receptor coupled to a G-protein to activate phospholipase C pathway, increasing the concentration of IP3 , which causes the release of Ca2+ from intracellular stores. The active constituents isolated from L. aspera namely, diterpenes, lignans and flavanoids can inhibit the action of prostaglandins (Sadhu et al., 2003, 2006). It was also reported that some of the anti-inflammatory effects produced by the flavanoids of L. aspera extract, viz., acacetin and apigenin were contributed by inhibiting the synthesis (as cyclooxygenase and 5-lipoxygenase inhibitors) of prostaglandins and leukotrienes, inhibition of PGE2 release and nitric oxide production. It appears that the extract produced a concentration dependant inhibition of fecundity by inhibiting the PGE2 mediated pathway. The present study indicated that L. aspera extract was highly effective in controlling hatching of eggs laid by the treated ticks. It was visually observed that the eggs laid by the treated ticks at all concentrations lacked the glossy
289
appearance that occurs due to waxy water proofing when compared to the eggs laid by control ticks. There could be two possibilities to explain the complete blocking of eclosion of eggs by extract of L. aspera: (a) decreased levels of ecdysteroids leading to decreased incorporation of free ecdysteroids in to the eggs. These free ecdysteroids are involved in oocyte maturation process. Friesen and Kaufman (2003) reported the inhibition of vitellogenesis and egg development in A. haebraeum by cypermethrin and attributed its cause as inhibition of release of 20-hydroxy ecdysone by the insecticide and (b) inhibition of PGE2 that may play a role in controlling the secretions of the tubular accessory sexual gland and the Gene organ in ticks thereby preventing the water proofing of the egg during passage through the vagina and outside. Proper wax coating is mandatory for proper hatching of eggs and maintenance of fecundity of ticks (Lees and Beament, 1946). It is also possible that the active substance/substances might interfere with the production of inhibitors of auto-oxidation of unsaturated egg wax lipids from the porose areas thereby desiccating the egg and killing the embryo. 4. Conclusion Ethanolic extract of L. aspera at very low concentration has a significant role in controlling reproduction of R. (B). annulatus ticks. Further studies to distinguish the active constituent which effectively blocks the eclosion process need to be undertaken. Acknowledgement Financial support from Indian Council of Agricultural Research through World Bank funded National Agricultural Innovation Project No. C2066 is thankfully acknowledged. References Aljamali, M.N., Bior, A.D., Sauer, J.R., Essenberg, R.C., 2003. RNA interference in ticks: a study using histamine binding protein ds RNA in the female tick Amblyomma americanum. Insect Mol. Biol. 12, 299–305. Binnington, K.C., Stone, B.F., 1977. Distribution of catecholamines in the cattle tick Boophilus microplus. Comp. Biochem. Physiol. 58C, 21–28. Bowman, A.S., Dillwith, J.W., Sauer, J.R., 1996. Tick salivary prostaglandins: presence, origin and significance. Parasitol. Today 12, 388–396. Chopra, R.N., Nayar, S.L., Chopra, I.C., 2002. Glossary of Indian Medicinal Plants. NISCAIR, CSIR, New Delhi, 153 pp. FAO, 1984. Ticks and Ticks Borne Disease Control. A Practical Field Manual. Vol. 1. Tick control, Rome. FAO, 2004. Guidelines Resistance Management and Integrated Parasite Control in Ruminants, Rome. Friesen, K.J., Kaufman, W.R., 2003. Cypermethrin inhibits egg development in the ixodid tick, Amblyomma hebraeum. Pestic. Biochem. Physiol. 76, 25–35. Goncalves, K., Toigo, E., Ascoli, B., von Poser, G., Ribeiro, V.S., 2007. Effects of solvents and surfactant agents on the female and larvae of cattle tick Boophilus microplus. Parasitol. Res. 100, 1267–1270. Ghosh, S., Azhahianambi, P., Yadav, M.P., 2007. Upcoming and future strategies of tick control: a review. J. Vector Borne Dis. 44, 79–89. 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. Jagannath, M.S., Muraleedharan, K., Hiregoudar, L.S., 1979. Prevalence of ixodid ticks of cattle at Bangalore. Indian J. Anim. Sci. 49, 890–894. Kaufman, W.R., Harris, R.A., 1983. Neural pathways mediating salivary fluid secretion in the ixodid tick Amblyomma hebraeum. Can. J. Zool. 61, 1976–1980.
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Koshy, T.J., Rajavelu, G., Lalitha, C.M., 1982. Ecology and bionomics of Boophilids of Tamil Nadu. Cheiron 11, 25–30. Lees, A.D., Beament, J.W.L., 1946. An egg—waxing organ in ticks. J. Cell Sci., 290–332. Mangathayaru, K., Thirumurugan, D., Patel, P.S., Pratap, D.V.V., David, D.J., Karthikeyan, J., 2006. Isolation and identification of nicotine from Leucas aspera (Willd) Link. Indian J. Pharm. Sci. 68, 88–90. Mwangi, R.W., Rembold, H., 1988. Growth inhibiting and larvicidal effects of Melia volkensii extracts on Aedes aegypti larvae. Entomol. Exp. Appl. 46, 103–108. Maheswaran, R., Sathish, S., Ignacimuthu, S., 2008. Larvicidal activity of Leucas aspera (Willd.) against the larvae of Culex quinquefasciatus Say. and Aedes aegypti L. Int. J. Integr. Biol. 2, 214–217. Muthukrishnan, J., Pushpalatha, H., Kasthuribhai, K., 1997. Biological effects of four plant extracts on Culex quinquefasciatus Say. Larval stages. Insect Sci. Appl. 17, 389–394. Nolon, J., 1990. Acaricide resistance in single and multi-host ticks and strategies for control. Parassitologia 32, 145–153. Rajamohanan, K., 1982. Identification of vector for babesiosis of cattle in Kerala. In: Proceedings of All India Symposium of Vectors and Vector Borne Diseases , Trivandrum, Kerala, pp. 125–128. Reddy, M.K., Viswananthan, S., Thirugnanasambatham, D., Santa, R., Lalitha, K., 1993a. Analgesic activity of Leucas aspera. Fitoterapia 64, 151–154.
Reddy, M.K., Viswananthan, S., Thirugnanasambatham, D., Santa, R., Lalitha, K., 1993b. Effect of Leucas aspera on snake venom poisoning in mice and its possible mechanism of action. Fitoterapia 64, 442–446. Rees, H.H., 2004. Hormonal control of tick development and reproduction. Parasitology 129, S127–S143. Roberts, L.S., Janovy, J., 2005. Foundation of Parasitology, 7th ed. McGraw Hill, pp. 644–646. Sadhu, S.K., Okuyama, E., Fujimoto, H., Ishibashi, M., 2003. Separation of Leucas aspera, a medicinal plant of Bangladesh, guided by prostaglandin inhibitory and antioxidant activities. Chem. Pharm. Bull. 51, 595–598. Sadhu, S.K., Okuyama, E., Fujimoto, H., Ishibashi, M., 2006. Diterpenes from Leucas aspera inhibiting prostaglandin induced contractions. J. Nat. Prod. 69, 988–994. Sankhon, N., Lockey, T., Rosell, R.C., Rothschild, M., Coons, L., 1999. Effect of methoprene and 20-hydroxyecdysone on vitellogenin production in cultured fat bodies and backless explants from unfed female Dermacentor variabilis. J. Insect Physiol. 45, 755–761. Wall, R., Shearer, D., 1997. Veterinary Entomology. Chappman and Hall, London, pp. 97–140. Weiss, B.L., Kaufman, W.R., 2001. The relationship between ‘critical weight’ and 20-hydroxyecdysone in the female ixodid tick, Amblyomma hebraeum. J. Insect Physiol. 47, 1261–1267.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Eclosion blocking effect of ethanolic extract of Leucas aspera (Lamiaceae) on Rhipicephalus (Boophilus) annulatus Reghu Ravindran a,∗ , Sanis Juliet b , A.R. Sunil a , K.G. Ajith Kumar a , Suresh N. Nair b , K.K. Amithamol a , M. Shynu c , Ajay Kumar Singh Rawat d , Srikanta Ghosh e a b c d e
Department of Veterinary Parasitology, College of Veterinary and Animal Sciences, Pookot, Wayanad, Kerala 673 576, India Department of Veterinary Pharmacology and Toxicology, College of Veterinary and Animal Sciences, Pookot, Wayanad, Kerala 673 576, India Department of Veterinary Biochemistry, College of Veterinary and Animal Sciences, Pookot, Wayanad, Kerala 673 576, India Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute, Lucknow UP226 001, India Division of Parasitology, Indian Veterinary Research Institute, Izatnagar UP243122, India
a r t i c l e
i n f o
Article history: Received 16 August 2010 Received in revised form 23 February 2011 Accepted 28 February 2011 Keywords: Leucas aspera Acaricidal activity Eclosion Rhipicephalus (Boophilus) annulatus
a b s t r a c t The crude ethanolic extract of aerial parts of Leucas aspera was tested for its acaricidal properties against Rhipicephalus (Boophilus) annulatus. The per cent adult mortality, inhibition of fecundity and hatching of laid ova were studied at concentrations of 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mg/ml. Adult tick mortality was significant at the highest concentration tested. Inhibition of fecundity of treated groups differed significantly from control and was concentration dependent. L. aspera extract also produced complete failure of eclosion of eggs from the treated ticks even at lower dilutions of the extract. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Ticks and the diseases they transmit are widely distributed throughout the world, particularly in tropical and subtropical regions. It has been estimated that 80 per cent of the world’s cattle population is exposed to tick infestation (FAO, 1984). The inherent problems of chemical acaricides like the resistance (Nolon, 1990) and environmental residues resulted in only partial success of tick control. The searches for environmentally safe products have accelerated the research on botanical acaricides. The active ingredients from plants are known to possess insecticidal, growth inhibiting, anti-moulting and repellent activities (Ghosh et al., 2007). Rhipicephalus (Boophilus) annulatus is a one host tick under subgenus Boophilus within the genus Rhipicephalus
∗ Corresponding author. Tel.: +91 9447713422; fax: +91 4936 256390. E-mail address: [email protected] (R. Ravindran). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.02.021
and occurs in greatest abundance in tropical and subtropical regions of the world. This cattle tick not only affects production but also acts as the vector of many protozoal, viral and rickettsial diseases (Wall and Shearer, 1997; Roberts and Janovy, 2005). Even though Rhipicephalus (Boophilus) microplus is the major tick affecting cattle of northern parts of India, R. (B.) annulatus is the commonest species found in southern India (Jagannath et al., 1979; Koshy et al., 1982; Rajamohanan, 1982). Leucas aspera (Lamiaceae) is a small herbaceous, erect plant with a free blooming nature that grows as a weed on waste lands and roadsides all over India. It is pungently aromatic and commonly used as antipyretic herb in south India. In traditional medicine it is indicated for cold, cough, painful swellings and chronic skin eruptions (Chopra et al., 2002). The plant was previously evaluated pharmacologically for its anti-inflammatory, analgesic and protective effects against cobra venom poisoning (Reddy et al., 1993a,b). Recently, Maheswaran et al. (2008) reported the larvicidal activity of the hexane extract of L. aspera against Culex quinquefasciatus and Aedes aegypti.
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R. Ravindran et al. / Veterinary Parasitology 179 (2011) 287–290
Table 1 Effects of different dilutions of ethanolic extract of L. aspera against R. (B.) annulatus. Sl no.
Concentration (mg/ml)
Mean tick weight per replicate ± SEM
1 2 3 4 5 6 7 8
Control 100 50 25 12.5 6.25 3.13 1.56
1.0182 1.0657 1.0665 1.0619 1.0736 1.0736 1.0098 0.9971
± ± ± ± ± ± ± ±
0.0382a 0.021a 0.0419a 0.0231a 0.0365a 0.0365a 0.0588a 0.0119a
Mean % adult mortality within 15 days ± SEM 0 54.16 45.83 8.33 4.16 4.16 4.16 4.16
± ± ± ± ± ± ± ±
0a 7.978b 7.978b 4.809a 4.165a 4.165a 4.165a 4.165a
Mean eggs mass per replicate ± SEM 0.4381 0.1419 0.1868 0.2601 0.2920 0.3018 0.3098 0.3280
± ± ± ± ± ± ± ±
0.0301d 0.0286a 0.0193ab 0.0371bc 0.0151c 0.0103c 0.0362c 0.0351c
Index of fecundity ± SEM
0.04299 0.1317 0.1753 0.2451 0.2757 0.2831 0.3049 0.3301
± ± ± ± ± ± ± ±
0.024d 0.0268a 0.0181ab 0.0370bc 0.0173c 0.0196c 0.0269c 0.0388c
Percentage inhibition of fecundity (%)
Hatching % (visual)
0 69.36 59.22 42.99 35.87 34.15 29.08 23.21
100 0 0 0 0 0 0 0
n = 4. Values are mean ± SEM, means bearing different superscripts a–d (P < 0.05), indicate significant difference when compared with the control.
The present study evaluates the potential of an ethanolic extract of L. aspera, as an acaricide against R. (B.) annulatus. 2. Materials and methods 2.1. Preparation of plant extract The L. aspera (Locally known as ‘Thumba’) were collected from Vythiri taluk of Wayanad district, Kerala. The voucher specimen was deposited in the herbarium of the National Botanical Research Institute, Lucknow. The aerial parts of the plant were cleaned and dried in shade at room temperature for two weeks. Dried plant material (1 kg) was pulverized using a pulverizer. The powdered plant (100 g) was used for ethanolic extraction in a soxhlet apparatus attached with solvent recovery unit (Rotavac, Buchi, Switzerland). Crude extract was dried at room temperature, weighed and dissolved in methanol for making the following dilutions of 1.56 mg/ml, 3.13 mg/ml, 6.25 mg/ml, 12.5 mg/ml, 25 mg/ml, 50 mg/ml and 100 mg/ml that were used for the testing. 2.2. Collection of ticks The fully engorged adult female ticks were collected from infested animals. The ticks were washed in tap water and dried on an absorbent paper. They were used for the adult immersion test (AIT). 2.3. In vitro acaricidal effects A total of 192 adult engorged female ticks were used for the present study. They were divided into eight groups (seven treatments and one control) each comprising of 24 ticks as six ticks each in four replicates. Each group was used to estimate the acaricidal effects of respective concentration of plant extract. The weight of each replicate of treated ticks was determined and immersed them for 2 min in 10 ml of the respective concentration of ethanolic extract. The control group was immersed in methanol. Ticks were recovered from the respective solution. They were dried using filter paper. Ticks of each replicate were placed in a separate specimen tube. They were incubated at 28 ◦ C and 80 per cent relative humidity (RH) in a BOD incubator. These ticks were observed for oviposition and death
up to 15 days. The per cent adult tick mortality and the weight of the eggs laid by the treated ticks were recorded in comparison with the control. The eggs were incubated at the same condition and the percentage of hatched eggs was estimated visually. The index of egg laying and percentage inhibition of fecundity were calculated using the following formulae (1) and (2), respectively (FAO, 2004; Goncalves et al., 2007): Index of egg laying (IE) =
weight of eggs laid (mg) weight of females (mg)
(1)
Percentage inhibition of fecundity (IF) (%) =
[IE (control group) − IE (treated group)] × 100 IE (control group)
(2)
2.4. Statistical analyses All the data were expressed as the mean ± SEM. Groups were compared using one-way ANOVA for repeated measurements using SPSS software. Duncan’s test was used for post hoc analysis. A value of P < 0.05 was considered significant. 3. Results and discussion The results of adult immersion test using the ethanolic extract of L. aspera are shown in Table 1. The efficacy of extract against R. (B.) annulatus females was assessed by estimating the per cent adult mortality, inhibition of fecundity and hatching rate. The per cent adult tick mortality caused by the ethanolic extract of the L. aspera varied from 4.16 to 54.16 per cent, when tested at concentrations ranging from 1.56 to 100 mg/ml. A statistically significant effect on adult mortality could be observed at higher concentrations of 50 mg/ml and 100 mg/ml. L. aspera has been used as an insecticide in traditional medicine in India. The larvicidal activity of L. aspera against mosquitoes was previously reported (Muthukrishnan et al., 1997; Maheswaran et al., 2008). They found first and second instar larvae of C. quinquefasciatus as more sensitive compared to third and four instars. Mwangi and Rembold (1988), reported that leaves of L. aspera exhibited heavy mortality especially during moulting process or subsequent process of melanization and tanning. Mangathayaru
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et al. (2006) isolated a liquid alkaloid from L. aspera and identified it as nicotine and speculated that the insecticidal property of the herb could be due to the presence nicotine. The results of the present study further demonstrated that the ethanolic extract of L. aspera induced a significant concentration dependant decrease in egg mass production. The inhibition of fecundity ranged from 23 to 69 per cent on the treated ticks. In ticks, the moulting hormone (ecdysteroids) plays a role in the regulation of salivary gland function, production of pheromones and oogenesis and oviposition (Rees, 2004). In fully engorged adult female ticks, the level of ecdysteroids rises in the haemolymph which in turn causes salivary gland to degenerate and as a result the ticks lay more eggs. This hormone also triggers vitellogenesis (Sankhon et al., 1999) and inhibits reattachment to the host (Weiss and Kaufman, 2001). The neuronal involvement in the control of salivary gland degeneration in the ixodid tick, Amblyomma haebraeum has been proved (Harris and Kaufman, 1984). The neurotransmitter dopamine regulates the synthesis of ecdysteroids. Dopamine was identified in the salivary gland and salivary gland nerves of R. (B.) microplus and A. haebraeum (Binnington and Stone, 1977; Kaufman and Harris, 1983). Dopamine binds to the D1 like receptor on the salivary glands to effect fluid secretion and synthesis of ecdysteroids. The dopamine receptor is coupled to a G-protein activated adenylate cyclase and the activation produces an increase in cAMP. The increase in cAMP regulates many aspects of cellular functions by activation of protein kinases. The protein kinases by catalyzing the phosphorylation of serine and threonine residues activate the target enzymes that synthesize ecdysteroids. The dopamine also opens a voltage gated Ca2+ channel allowing influx of extracellular Ca2+ to stimulate cytosolic phospholipase A2 to liberate arachidonic acid. This arachidonic acid is then converted by a cyclooxygenase pathway to Prostaglandin E2 (PGE2 ) and Prostaglandin F2␣ (PGF2␣ ) and small amounts of Prostaglandin I2 (PGI2 ) (Aljamali et al., 2003). PGE2 and PGF2␣ are found in very high concentration in tick saliva and salivary glands (Bowman et al., 1996). In addition, PGE2 plays a very important role in peristaltic activity of oviduct to transport the oocytes to the genital aperture. It interacts with the Eicosapentenoic acid (EP1 ) like receptor coupled to a G-protein to activate phospholipase C pathway, increasing the concentration of IP3 , which causes the release of Ca2+ from intracellular stores. The active constituents isolated from L. aspera namely, diterpenes, lignans and flavanoids can inhibit the action of prostaglandins (Sadhu et al., 2003, 2006). It was also reported that some of the anti-inflammatory effects produced by the flavanoids of L. aspera extract, viz., acacetin and apigenin were contributed by inhibiting the synthesis (as cyclooxygenase and 5-lipoxygenase inhibitors) of prostaglandins and leukotrienes, inhibition of PGE2 release and nitric oxide production. It appears that the extract produced a concentration dependant inhibition of fecundity by inhibiting the PGE2 mediated pathway. The present study indicated that L. aspera extract was highly effective in controlling hatching of eggs laid by the treated ticks. It was visually observed that the eggs laid by the treated ticks at all concentrations lacked the glossy
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appearance that occurs due to waxy water proofing when compared to the eggs laid by control ticks. There could be two possibilities to explain the complete blocking of eclosion of eggs by extract of L. aspera: (a) decreased levels of ecdysteroids leading to decreased incorporation of free ecdysteroids in to the eggs. These free ecdysteroids are involved in oocyte maturation process. Friesen and Kaufman (2003) reported the inhibition of vitellogenesis and egg development in A. haebraeum by cypermethrin and attributed its cause as inhibition of release of 20-hydroxy ecdysone by the insecticide and (b) inhibition of PGE2 that may play a role in controlling the secretions of the tubular accessory sexual gland and the Gene organ in ticks thereby preventing the water proofing of the egg during passage through the vagina and outside. Proper wax coating is mandatory for proper hatching of eggs and maintenance of fecundity of ticks (Lees and Beament, 1946). It is also possible that the active substance/substances might interfere with the production of inhibitors of auto-oxidation of unsaturated egg wax lipids from the porose areas thereby desiccating the egg and killing the embryo. 4. Conclusion Ethanolic extract of L. aspera at very low concentration has a significant role in controlling reproduction of R. (B). annulatus ticks. Further studies to distinguish the active constituent which effectively blocks the eclosion process need to be undertaken. Acknowledgement Financial support from Indian Council of Agricultural Research through World Bank funded National Agricultural Innovation Project No. C2066 is thankfully acknowledged. References Aljamali, M.N., Bior, A.D., Sauer, J.R., Essenberg, R.C., 2003. RNA interference in ticks: a study using histamine binding protein ds RNA in the female tick Amblyomma americanum. Insect Mol. Biol. 12, 299–305. Binnington, K.C., Stone, B.F., 1977. Distribution of catecholamines in the cattle tick Boophilus microplus. Comp. Biochem. Physiol. 58C, 21–28. Bowman, A.S., Dillwith, J.W., Sauer, J.R., 1996. Tick salivary prostaglandins: presence, origin and significance. Parasitol. Today 12, 388–396. Chopra, R.N., Nayar, S.L., Chopra, I.C., 2002. Glossary of Indian Medicinal Plants. NISCAIR, CSIR, New Delhi, 153 pp. FAO, 1984. Ticks and Ticks Borne Disease Control. A Practical Field Manual. Vol. 1. Tick control, Rome. FAO, 2004. Guidelines Resistance Management and Integrated Parasite Control in Ruminants, Rome. Friesen, K.J., Kaufman, W.R., 2003. Cypermethrin inhibits egg development in the ixodid tick, Amblyomma hebraeum. Pestic. Biochem. Physiol. 76, 25–35. Goncalves, K., Toigo, E., Ascoli, B., von Poser, G., Ribeiro, V.S., 2007. Effects of solvents and surfactant agents on the female and larvae of cattle tick Boophilus microplus. Parasitol. Res. 100, 1267–1270. Ghosh, S., Azhahianambi, P., Yadav, M.P., 2007. Upcoming and future strategies of tick control: a review. J. Vector Borne Dis. 44, 79–89. 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. Jagannath, M.S., Muraleedharan, K., Hiregoudar, L.S., 1979. Prevalence of ixodid ticks of cattle at Bangalore. Indian J. Anim. Sci. 49, 890–894. Kaufman, W.R., Harris, R.A., 1983. Neural pathways mediating salivary fluid secretion in the ixodid tick Amblyomma hebraeum. Can. J. Zool. 61, 1976–1980.
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