Acaricidal Activity Of Plant Extracts And Essential Oil And Their Isolated Compounds Against Haemaphysalis Longicornis

The tick Haemaphysalis longicornis(Neumann)is a well-known vector of numerous pathogens of veterinary and medical importance.Although synthetic acaricides including pyrethroids represent the main strategy in controlling ticks including H.longicornis,the unintended effects on the environment and non-target organisms associated with their intensive and injudicious use is a major challenge to their continued usage.Consequently,there is a need for an effective and environmentally friendly alternative methods,one of which is the use of plant essential oil(EO)and/or extract.However,EOs and/or extracts are usually complex mixtures of volatile compounds,and these compounds can vary in composition as a result of biotic and abiotic factors.Thus,understanding the contribution of the individual compounds particularly their interactions with respect to acaricidal activity is important for the use of EO and/or extract as an alternative to synthetic acaricides.In the present study,the potential of EO and crude extract as acaricides for H.longicornis control was evaluated.The crude methanolic extracts of Cinnamomum cassia,Pogostemon cablin,and Sophora flavescens were initially screened against the unfed adults of H.longicornis using a topical application,and the most effective crude extract was further evaluated for acaricidal activity against the engorged females of H.longicornis using the adult immersion test(AIT).The results indicated that C.cassia crude extract demonstrated strong acaricidal activity(93.00%mortality)compared with the other crude extracts(0.00%mortality)at 20%concentration after 24 h of topical application.Results obtained using AIT revealed the acaricidal activity of C.cassia extract against H.longicornis engorged adult females.Inhibition of oviposition was 12.70%at the lowest concentration(1.25%)and reached 98.06%at the highest concentration(20%).Treatment with 20%concentration resulted in complete inhibition of larval hatching.Thus,C.cassia was used for subsequent tests.In the subsequent test,C.cassia crude extract and EO were studied.The acaricidal activity of the extract and EO,and their major components determined by gas chromatography-mass spectrometry were evaluated against the unfed larvae and nymphs of H.longicornis using the larval and nymphal packet test.The most effective substance(s)was further evaluated for acaricidal activity against the engorged larvae and nymphs using the immersion test and for feeding deterrent property against the unfed nymphs of this tick(in vivo).According to the results,(E)-cinnamaldehyde was the predominant component of the crude extract(50.79%)and EO(89.95%).The 50%lethal concentration(LC₅₀)for the unfed larvae and nymphs treated with the crude extract was 11.56 and 49.18 mg/m L after 24 h of LPT and NPT,respectively.The EO,(E)-cinnamaldehyde,and fenvalerate(positive control)exhibited acaricidal activity,with LC₅₀ values of 3.81,3.15,and 0.14 mg/m L,respectively,against the larvae,and21.31,16.93,and 1.89 mg/m L,respectively,against the nymphs after 24 h.The results also revealed that the EO,(E)-cinnamaldehyde,and fenvalerate showed acaricidal activity against the engorged larvae and nymphs of H.longicornis,with LC₅₀ values of 1.46 and 16.69 mg/m L,3.86 and 17.96 mg/m L,and 0.06 and 5.05 mg/m L,respectively after 25 days.In the feeding deterrent test,(E)-cinnamaldehyde displayed feeding deterrent activity against H.longicornis nymphs at a concentration of 33.86mg/m L.This study also expands on previous EO-based acaricide research.Specifically,understanding the contribution of the individual constituents of cinnamon(Cinnamomum)EO to its overall toxicity and how these constituents interact to give a joint effect when present in a mixture with respect to acaricidal activity.Herein,the acaricidal activity of constituents from EOs of cinnamon(C.cassia and Cinnamomum zeylanicum)against H.longicornis was investigated using the nymphal and adult immersion tests.Among the test compounds,benzyl benzoate showed the highest toxicity against the nymphs(LC₅₀=24.41 mg/m L)and adults(LC₅₀=3.99 mg/m L).All combinations at their sublethal concentration(LC₃₀)exhibited synergy against H.longicornis adults.The binary mixture of benzyl benzoate+cinnamaldehyde presented the highest synergy ratio(3.42)while tertiary mixtures of cinnamaldehyde+eugenol+linalool showed the least synergy ratio(1.20).Furthermore,biochemical assays of H.longicornis larvae and nymphs revealed that esterase,monooxygenase,glutathione-S-transferase(GST),acetylcholinesterase(ACh E),and Na⁺/K⁺-adenosine triphosphatases(ATPase)activities were significantly(p<0.05)affected either by C.cassia EO or(E)-cinnamaldehyde depending on the exposure time.Benzyl benzoate significantly(p<0.05)inhibited ACh E activity of H.longicornis adults.Molecular analysis results indicated that C.cassia EO and(E)-cinnamaldehyde treatment significantly(p<0.05)increased adenosine triphosphate(ATP)-binding cassette(ABC)transporters(ABCB10)gene expression in H.longicornis larvae.Finally,the potential effects of C.cassia EO,(E)-cinnamaldehyde,and benzyl benzoate on non-target species were investigated using Tenebrio molitor L and Harmonia axyridis Pallas as representatives of terrestrial organisms and the water flea,Daphnia magna Straus as representative of aquatic organism.The results revealed that topical application of C.cassia EO(tested concentration:LC₅₀=3.81 and 21.31 mg/m L for unfed larvae and nymphs of H.longicornis)or(E)-cinnamaldehyde(tested concentration:LC₅₀=3.15 and 16.93 mg/m L,respectively)did not cause mortality in T.molitor or H.axyridis adults.However,topical application of fenvalerate(tested concentration:LC₅₀=1.89 mg/m L for the nymphs of H.longicornis)resulted in significant mortality in adult T.molitor(18.00±7.35)and H.axyridis(8.00±3.74).In addition,spray application of cinnamaldehyde at a concentration two times greater than the LC₅₀(2 x LC₅₀=28.34)estimated against the adults of H.longicornis caused significant mortality of T.molitor adults(16.67%)compared with the negative control.However,mortality was significantly lower when compared with the positive control(63.33%)at 2 x LC₅₀=0.002.Furthermore,no mortality was recorded in the adults of T.molitor treated with cinnamaldehyde at LC₅₀(28.34)and1/2of LC₅₀,and with benzyl benzoate at the various concentrations(i.e.,LC₅₀=3.99,1/2of LC₅₀ and 2 x LC₅₀).On the other hand,application of cinnamaldehyde at a sublethal concentration(1/2of LC₅₀=28.34)resulted in significant mortality(100%)of D.magma adults,compared with the application of benzyl benzoate(12.5%)(at a sublethal concentration of1/2of LC50=3.99)at 24 h post-treatment.However,the sublethal concentration of benzyl benzoate resulted in 99.75%mortality of this non-target aquatic organism at 48 h post-treatment.In conclusion,this study demonstrates that C.cassia EO,crude extract and(E)-cinnamaldehyde have the potential to be developed into botanical-based acaricides for the control of H.longicornis.However,the high mortality of non-target aquatic organisms exposed to cinnamon EO components may be a major challenge in the development of potential plant acaricides based on this EO.Furthermore,the acaricidal activity of cinnamon EO against H.longicornis may be linked to the content of benzyl benzoate whose presence may be synergistic to the activity of other compounds including(E)-cinnamaldehyde.