Repellent effects of Chinese cinnamon oil on nymphal ticks of Haemaphysalis longicornis, Rhipicephalus haemaphysaloides, and Hyalomma asiaticum.

The repellent activity of Chinese cinnamon oil (Cinnamomum cassia) on nymphal ticks (Haemaphysalis longicornis Neumann, Rhipicephalus haemaphysaloides Supino, and Hyalomma asiaticum Schulze and Schlottke) was evaluated in a sample Y-tube bioassay. The results were based on the vertical migration of ticks during the host-seek phase and showed a dose-dependent repellent effect of Chinese cinnamon oil on the tested nymphs after 6 h. For H. longicornis, R. haemaphysaloides, and H. asiaticum at the concentrations (vol/vol) of 3, 3, and 1.5%, the repellent percentages over time were 68-97, 69-94, and 69-93%, respectively, which indicated strong repellent activities against ticks, similar to the positive control DEET (N,N-diethyl-3-methylbenzamide). Chinese cinnamon oil exerted the strongest effect on H. asiaticum nymphs. To our knowledge, this is the first study to investigate the repellent effects of Chinese cinnamon oil on ticks. Chinese cinnamon oil has considerable potential and should be developed as a practical tick repellent.

Acaricidal activity of Cinnamomum cassia (Chinese cinnamon) against the tick Haemaphysalis longicornis is linked to its content of (E)-cinnamaldehyde.

BACKGROUND: The tick Haemaphysalis longicornis (Neumann) is a well-known vector of numerous pathogens of veterinary and medical importance. Various control strategies, including the use of synthetic pesticides, have been developed to control this tick species. However, demand for effective and safe alternative pesticides is increasing due to the adverse effects associated with the intensive and injudicious use of synthetic pesticides, which include undesirable effects on non-target species and environmental pollution. Hence, the acaricidal activity of the extract and the essential oil of Cinnamomum cassia (Chinese cinnamon) and their major components, and the underlying mechanisms of this activity, were evaluated against unfed larvae and nymphs of H. longicornis. METHODS: The components of the extract and essential oil of C. cassia were determined by gas chromatography-mass spectrometry, and their larvicidal and nymphicidal activity were evaluated using the larval and nymphal packet test. The underlying detoxification mechanism was elucidated by targeting in vivo esterase and monooxygenase activity, and the toxicological effect was assessed on non-target Tenebrio molitor and Harmonia axyridis by topical application in open Petri dishes. RESULTS: (E)-cinnamaldehyde was the predominant component of the extract (50.79%) and essential oil (89.95%). The 50% lethal concentration (LC50) for larvae and nymphs treated with the extract was 11.56 and 49.18 mg/mL, respectively. The essential oil, (E)-cinnamaldehyde and fenvalerate exhibited acaricidal activity, with LC50 values of 3.81, 3.15, and 0.14 mg/mL, respectively, against the larvae, and 21.31, 16.93, and 1.89 mg/mL, respectively, against the nymphs. (E)-cinnamaldehyde significantly increased esterase and monooxygenase activity in both larvae and nymphs. Unlike fenvalerate, C. cassia essential oil and (E)-cinnamaldehyde did not cause mortality of T. molitor or H. axyridis adults. CONCLUSIONS: This study demonstrates that C. cassia essential oil and (E)-cinnamaldehyde have the potential to be developed into botanical-based larvicidal and nymphicidal agents for tick control.

Activity of the extract of Acmella oleracea on immature stages of Amblyomma sculptum (Acari: Ixodidae).

This study evaluated the acaricidal activity of the methanol extract of Acmella oleracea with 0.187% of spilanthol against immature stages of Amblyomma sculptum. The packet test was used to evaluate the extract's activity on unengorged larvae and nymphs, testing concentrations of 0.4 to 50 mg/mL for larvae and 12.5 to 200.0 mg/mL for nymphs. For the engorged stages, the immersion test was used, at concentrations of 0.4 to 50 mg/mL for larvae and 12.5 to 200.0 mg/mL for nymphs. The methanol extract caused 100% mortality of the unengorged larvae and nymphs starting at concentrations of 12.5 and 200.0 mg/mL, respectively. For engorged larvae and nymphs, the mortality was 100% starting from concentrations of 12.5 and 150.0 mg/mL, respectively. The LC50 for unengorged larvae was 3.2 mg/mL, while for engorged larvae it was 6.6 mg/mL. For unengorged nymphs, the LC50 was 38.5 mg/mL, but it was not possible to calculate the corresponding value for engorged nymphs because the data did not fit the probit model. These results demonstrate that the methanol extract of A. oleracea has acaricidal activity against different immature stages of A. sculptum.

Repellency to ticks (Acari: Ixodidae) of extracts of Nigella sativa (Ranunculaceae) and the anti-inflammatory DogsBestFriend™.

Motivated by observations that the canine anti-inflammatory cream DogsBestFriend™ (DBF) appeared to deter flies, mosquitoes, and ticks from treated animals, repellent efficacy bioassays using four species of ticks were conducted with three extracts of Nigella sativa L. (Ranunculaceae), a constituent of DBF. The DBF cream was tested against nymphs of lone star tick, Amblyomma americanum (L.). In vertical filter paper assays, the three extracts applied at 0.413 mg extract/cm(2) filter paper repelled 96.7-100 % of brown dog tick, Rhipicephalus sanguineus (Latreille) nymphs, whereas, at the same rate, only one extract repelled >90 % A. americanum nymphs. Adult (mixed sexes) American dog ticks, Dermacentor variabilis (Say), required a higher concentration to be repelled effectively; two extracts, applied at 0.827 mg extract/cm(2) filter paper, repelled ≥90 % of the D. variabilis. In contrast, all extracts applied at much lower concentration (0.206 mg extract/cm(2) filter paper) repelled 100 % adult blacklegged ticks, Ixodes scapularis Say (only females tested). Of the two more repellent extracts, one lost most of its activity against A. americanum nymphs in <4 h when applied at 0.827 mg extract/cm(2) filter paper, whereas the other repelled 66.7 % of the nymphs at 192 h after application. At 0.206 mg extract/cm(2) filter paper, one extract was as repellent as deet against A. americanum nymphs. In a vertical bioassay in which nylon organdy was substituted for filter paper, DBF, at the rates of 1.67 and 0.835 mg cream/cm(2), repelled 76.7 and 30.0 % A. americanum nymphs, respectively. These findings indicate that when applied appropriately DBF should afford some protection to canines against tick bites.

Assessment of the repellent effect of Lippia alba essential oil and major monoterpenes on the cattle tick Rhipicephalus microplus.

The control of Rhipicephalus microplus (Ixodida: Ixodidae) is achieved using synthetic acaricides. However, resistant tick populations are widespread around the world. Plant essential oils can act as repellents, keeping ticks away from hosts and decreasing the selection pressure on synthetic acaricides. The aim of this study was to evaluate the in vitro repellent effect of Lippia alba essential oil on R. microplus larvae. Leaves from two L. alba genotypes maintained under the same agronomic and environmental conditions were collected. Essential oil was extracted by hydrodistillation and analysed by gas chromatography-mass spectrometry (GC-MS). The major monoterpenes detected in the chemical analysis were commercially acquired and tested. For the repellency test, a glass rod was vertically fixed to measure active climbing of approximately 30 R. microplus larvae aged 14-21 days in response to essential oils and monoterpenes. Repellency was evaluated at 1 h, 3 h and 5 h after treatment. Variation in repellent action was detected between the genotypes. The major monoterpenes identified in the essential oils (limonene and carvone) showed low repellent effects in comparison with intact essential oils. Thus, the present results showed that L. alba essential oil contains bioactive compounds with great repellent activity against ticks that varies according to the plant genotype.

Acaricidal activity of synthesized titanium dioxide nanoparticles using Calotropis gigantea against Rhipicephalus microplus and Haemaphysalis bispinosa.

OBJECTIVE: To assess the acaricidal activity of titanium dioxide nanoparticles (TiO2 NPs) synthesized from flower aqueous extract of Calotropis gigantea(C. gigantea) against the larvae of Rhipicephalus (Boophilus) microplus [R. (B.) microplus] and the adult of Haemaphysalis bispinosa (H. bispinosa). METHODS: The lyophilized C. gigantea flower aqueous extract of 50 mg was added with 100 mL of TiO(OH)2 (10 mM) and magnetically stirred for 6 h. Synthesized TiO2 NPs were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), and Energy dispersive X-ray spectroscopy (EDX). The synthesised TiO2 NPs were tested against the larvae of R. (B.) microplus and adult of H. bispinosa were exposed to filter paper impregnated method. RESULTS: XRD confirmed the crystalline nature of the nanoparticles with the mean size of 10.52 nm. The functional groups for synthesized TiO2 NPs were 1 405.19, and 1 053.45 cm(-1) for -NH2 bending, primary amines and amides and 1 053.84 and 1 078.45 cm(-1) for C-O. SEM micrographs of the synthesized TiO2 NPs showed the aggregated and spherical in shape. The maximum efficacy was observed in the aqueous flower extract of C. gigantea and synthesized TiO2 NPs against R. (B.) microplus (LC50=24.63 and 5.43 mg/L and r(2)=0.960 and 0.988) and against H. bispinosa (LC50= 35.22 and 9.15 mg/L and r(2) = 0.969 and 0.969), respectively. CONCLUSIONS: The synthesized TiO2 NPs were highly stable and had significant acaricidal activity against the larvae of R. (B.) microplus and adult of H. bispinosa. This study provides the first report of synthesized TiO2 NPs and possessed excellent anti-parasitic activity.

Multiple ferritins are vital to successful blood feeding and reproduction of the hard tick Haemaphysalis longicornis.

Ticks are obligate hematophagous parasites and important vectors of diseases. The large amount of blood they consume contains great quantities of iron, an essential but also toxic element. The function of ferritin, an iron storage protein, and iron metabolism in ticks need to be further elucidated. Here, we investigated the function a newly identified secreted ferritin from the hard tick Haemaphysalis longicornis (HlFER2), together with the previously identified intracellular ferritin (HlFER1). Recombinant ferritins, expressed in Escherichia coli, were used for anti-serum preparation and were also assayed for iron-binding activity. RT-PCR and western blot analyses of different organs and developmental stages of the tick during blood feeding were performed. The localization of ferritins in different organs was demonstrated through an indirect immunofluorescent antibody test. RNA interference (RNAi) was performed to evaluate the importance of ferritin in blood feeding and reproduction of ticks. The midgut was also examined after RNAi using light and transmission electron microscopy. RT-PCR showed differences in gene expression in some organs and developmental stages. Interestingly, only HlFER2 was detected in the ovary during oviposition and in the egg despite the low mRNA transcript. RNAi induced a reduction in post-blood meal body weight, high mortality and decreased fecundity. The expression of vitellogenin genes was affected by silencing of ferritin. Abnormalities in digestive cells, including disrupted microvilli, and alteration of digestive activity were also observed. Taken altogether, our results show that the iron storage and protective functions of ferritin are crucial to successful blood feeding and reproduction of H. longicornis.

Acaricidal effect of essential oils from Lippia graveolens (Lamiales: Verbenaceae), Rosmarinus officinalis (Lamiales: Lamiaceae), and Allium sativum (Liliales: Liliaceae) against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

Acaricidal effects of three essential oils extracted from Mexican oregano leaves (Lippia graveolens Kunth), rosemary leaves (Rosmarinus officinalis L.), and garlic bulbs (Allium sativum L.) on 10-d-old Rhipicephalus (Boophilus) microplus (Canestrini) tick larvae were evaluated by using the larval packet test bioassay. Serial dilutions of the three essential oils were tested from a starting concentration of 20 to 1.25%. Results showed that both Mexican oregano and garlic essential oils had very similar activity, producing high mortality (90-100%) in all tested concentrations on 10-d-old R. microplus tick larvae. Rosemary essential oil produced >85% larval mortality at the higher concentrations (10 and 20%), but the effect decreased noticeably to 40% at an oil concentration of 5%, and mortality was absent at 2.5 and 1.25% of the essential oil concentration. Chemical composition of the essential oils was elucidated by gas chromatography-mass spectrometry analyses. Mexican oregano essential oil included thymol (24.59%), carvacrol (24.54%), p-cymene (13.6%), and y-terpinene (7.43%) as its main compounds, whereas rosemary essential oil was rich in a-pinene (31.07%), verbenone (15.26%), and 1,8-cineol (14.2%), and garlic essential oil was rich in diallyl trisulfide (33.57%), diallyl disulfide (30.93%), and methyl allyl trisulfide (11.28%). These results suggest that Mexican oregano and garlic essential oils merit further investigation as components of alternative approaches for R. microplus tick control.

Repellent activity of plant-derived compounds against Amblyomma cajennense (Acari: Ixodidae) nymphs.

Repellence responses of Amblyomma cajennense nymphs to callicarpenal, intermedeol, Hyptis suaveolens essential oil, extract of Melia azedarach, Cymbopogon nardus, Spiranthera odoratissima, Chenopodium ambrosioides, Ageratum conyzoides, Mentha pulegium, Ruta graveolens, and Memora nodosa were studied. Among these the extract of C. nardus stood out because of the long-lasting repellence, maintaining, in the highest concentration, 35h of protection against 90% of the nymphs. The essential oil of H. suaveolens and the extracts of C. ambrosioides and A. conyzoides showed good repellence index (66%) when applied in high concentrations. However, greater protection could be obtained at higher concentrations but with a shorter repellence time. Callicarpenal, intermedeol, extract of M. Pulegium, and M. nodosa leaves showed moderate repellence in high concentrations. Extracts from M. azedarach, R. graveolens, S. odoratissima, and M. nodosa roots showed little or no repellent effect. These results show that some plant extracts may represent a promising alternative in the control of infestations by A. cajennense.

Sustained control of Gibson Island, Maryland, populations of Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) by community-administered 4-Poster deer self-treatment bait stations.

In 1998, twenty-five 4-Poster deer treatment bait stations were deployed on Gibson Island (GI), Maryland, as part of the U.S. Department of Agriculture (USDA) Northeast Area-Wide Tick Control Project. Treatments concluded in June 2002, having achieved 80% and 99.5% control of blacklegged ticks, Ixodes scapularis, and lone star ticks, Amblyomma americanum, respectively. No area-wide tick control was attempted again on the island until 2003, when 15 Dandux-manufactured 4-Posters were purchased by the GI Corporation and operated until the present. Annual flagging at sites on the island and a similar untreated area on the nearby mainland in May and June from 1998 to 2007 has demonstrated that populations of host-seeking nymphs of both tick species have remained at consistently low levels on the island during GI Corporation administration of the 4-Posters, in spite of 40% fewer 4-Posters and increased deer density during 2003-2007.

Cloning and characterization of a cDNA clone encoding calreticulin from Haemaphysalis qinghaiensis (Acari: Ixodidae).

The application of anti-tick vaccine has been shown to be the most promising alternative strategy compared to the current use of acaricides that suffer from a number of serious limitations. The success of this method is dependent upon identification and cloning of potential tick vaccine antigens. Previously, we have cloned 21 positive clones (named from Hq02 to Hq22) by immunoscreening complimentary DNA (cDNA) libraries of Haemaphysalis qinghaiensis; however, some of those clones did not contain open reading frames (ORF). In this study, we amplified the entire sequence of Hq07 by using rapid amplification of the cDNA ends. Hq07 contains an ORF of 1,233 bp that encodes for 410 amino acid residues with a coding capacity of 47 kDa. Search of the cloned sequences against GenBank revealed that Hq07 is a calreticulin (CRT)-similar clone and designated HqCRT. Expression analysis by reverse transcription-polymerase chain reaction showed that this gene is ubiquitously expressed at different developmental stages and in different tissues of H. qinghaiensis. The gene was expressed as glutathione S-transferase-fused proteins in a prokaryotic system. Western blot analysis revealed that native HqCRT was secreted into their hosts by ticks during blood sucking. Vaccination of sheep with rHqCRT conferred protective immunity against ticks, resulting in 54.3% mortality in adult ticks, compared to the 38.7% death rate in the control group. These results demonstrated that rHqCRT might be a useful vaccine candidate antigen for biological control of H. qinghaiensis.

The molecular and biological analysis of ixodid ticks histamine release factors.

We previously described a Dermacentor varibialis (DV) cDNA that encodes a ubiquitously expressed and tick saliva-secreted functional histamine release factor (HRF) homolog. In this study gene specific primers based on DVHRF open reading frame nucleotide sequence were utilized to amplify three orthologs, from the wood tick, D. andersoni (DA), the black legged tick, the southern cattle tick, Boophilus microplus (BM) and the lone star tick, Amblyomma americanum (AA). At nucleotide level, sequence comparisons revealed 98 89 and 84% similarity to DVHRF for DAHRF, AAHRF and BMHRF, respectively, while predicted polypeptide comparisons revealed 98, 96 and 91% similarity for DAHRF, AAHRF and BMHRF respectively. Phylogenetically, the tick HRF clade, while distinct (100% bootstrap value), is closely related to other arthropods, but distantly related to vertebrate and protozoan clades. Consistent with sequence similarity analysis, a DVHRF-specific northern blotting probe hybridized a approximately 900 base pair (bp) mRNA band on all RNA blots. Likewise a mouse polyclonal antibody to E. coli-expressed recombinant (r) DVHRF, cross-reacted baculovirus-expressed non-fusion rAAHRF, rDAHRF, and rBMHRF. As revealed by northern blotting analysis of larvae and nymph RNA, DVHRF mRNA is expressed in both immature and mature ticks indicating that its transcription is not developmentally regulated. Unlike rHRF/TCTP proteins of other organisms, the calcium-binding function may not be conserved for tick HRF homologs as revealed by the 45CaCl2+ overlay assay. Apparent global expression of DVHRF and its orthologs make this protein family an ideal target antigen for development of novel tick control strategies targeting multiple tick species.

Acaricidal effects of cardiac glycosides, azadirachtin and neem oil against the camel tick, Hyalomma dromedarii (Acari: Ixodidae).

The cardiac glycoside, digitoxin, from Digitalis purpurea L (Scrophulariaceae), a cardiac glycosidal (cardenolide) extract from Calotropis procera (Ait) R Br (Asclepiadaceae), azadirachtin and neem oil from Azadirachta indica A Juss (Meliaceae) were tested for their effects against larvae and adult stages of the camel tick, Hyalomma dromedarii Koch (Acari: Ixodidae). The contact LC50 values of the first three materials against adults were 4.08, 9.63 and >40.7 microg cm(-2), respectively, whereas the dipping LC50 values of the four materials were 409.9, 1096, >5000 and >5000 mg litre(-1), respectively. Contact and dipping LC50 values of the extract and azadirachtin against larvae were 6.16, >20.3 microg cm(-2) and 587.7 and >2500 mg litre(-1), respectively. Azadirachtin had no effects on egg production or feeding of adults up to 5000 mg litre(-1); however at 2500 mg litre(-1), it caused significant reduction in feeding activity of larve, prolonged the period for moulting to nymphal stage, and caused 60% reduction in moultability. Results of the two cardiac glycoside materials are comparable with those of several commercial acaricides. The risks and benefits associated with the use of cardiac glycosides are considered.

In vitro acaricidal effect of plant extract of neem seed oil (Azadirachta indica) on egg, immature, and adult stages of Hyalomma anatolicum excavatum (Ixodoidea: Ixodidae).

Effects of the plant extract of neem seed (Azadirachta indica) on eggs, immature, and adult stages of Hyalomma anatolicum excavatum was studied at concentrations of 1.6, 3.2, 6.4, and 12.8%. The extract was found to have a significant effect on the hatching rate of eggs. It significantly increased the hatching rate during the first 7 days post-treatment (DPT) giving incompletely developed and dead larvae; however, it cause hatching failure at DPT 15. Neem Azal F induced a significant increased in mortality rates of newly hatched larvae, unfed larvae, and unfed adults reaching 100% on 15th, 3rd, and 15th DPT, respectively. The mortality rates increased with the extract concentrations. Although, it had no significant effect on the moulting rates of fed nymphs, it caused malformation or deformities in 4% of adults moulted. It was concluded that the concentration of Neem Azal F which may be used for commercial control of this tick species were 1.6 and 3.2%.