Ticks

Dermacentor silvarum Olenev and the bisexual population of Haemaphysalis longicornis Neumann were collected from Zhangjiakou, Hebei (N 36°05′–42°40′, E 113°27′–119°50′). Haemaphysalis doenitzi Warburton et Nuttal and the parthenogenic population of H. longicornis were collected from Cangxi, Sichuan (N 31°37′–32°10′, E 105°43′–106°28′). Hyalomma asiaticum Schulze and Schlottke was collected from Urumqi, Xinjiang (N 34°22′–49°33′, E 73°41′–96°18′). All ticks were collected by dragging a white flannel cloth (1.2 m long × 1.0 m wide), stretched between two wooden rods, at a speed of 10 m/min for 500 m over vegetation. The clothes were checked every 20 m, and ticks attached to the cloth were collected, placed separately in 10-ml plastic tubes, and transported to the laboratory. After identification, the collected ticks were fed on rabbits. To facilitate feeding and management, ticks were placed into cloth bags attached by adhesive tape onto the ears of rabbits and checked daily (Liu et al. 2005). After engorgement, females were placed in separate 10-ml glass tubes for oviposition in a laboratory incubator maintained at 20 ± 1°C, 90% relative humidity, and a photoperiod of 16 h light, 8 h darkness. The use of rabbits for the purpose of this study was approved by the Animal Care and Use Committee of Hebei Normal University.

Egg treatments and wax extraction

Eggs were collected daily during the oviposition and stored at −20°C in a refrigerator until use in the assays. Wax was extracted from selected eggs of each tick species using a cholorform/methanol (v/v = 2:1) and distilled water extraction method modified from Yu et al. (2012). Simply, 1 g of tick eggs was placed in 3 ml chloroform/methanol in a glass tube and vortexed for 15 s; the supernatant was transferred to a clean glass tube. Then, the eggs were immersed in 1 ml distilled water and vortexed; the supernatant was transferred and combined with the previous supernatant. The eggs were washed with cholorform/methanol (v/v = 2:1) again, and that supernatant was combined with the previous supernatant. The combined supernatant was centrifuged at 1400 ×g for 10 min to separate the phases. The upper phase (aqueous extract) was lyophilized and stored at −80°C, and the lower phase (organic extract) was dried under a nitrogen stream in a fume hood and stored at −20°C until ready for use. After extraction, the eggs from which the wax had been extracted were stored.

Assays of antimicrobial activity associated with eggs and extracted wax

An in vitro agar diffusion assay was used to determine the antimicrobial activity associated with wax extracts from the tick eggs, eggs from which the waxes had been extracted, and eggs that were not subjected to was extraction as previously described by Yu et al. (2012). Cultures of the gram-positive bacteria Staphylococcus aureus Rosenbach (ATCC25923), Enterococcus faecalis (Andrewes and Horder) Schleifer and Kilpper-Balz (ATCC981), Enterococcus faecium (Orla-Jensen) Schleifer and Kilpper-Balz (ATCC091299), and Nocardia asteroides (Eppinger) Blanchard (ATCC201118) and the gram-negative bacteria Escherichia coli (Migula) Castellani and Chalmers (ATCC25922), Klebsiella pneumonia (Schroeter) Trevisan (ATCC43816), Salmonella enteritidis (Gaertner) Castellani and Chalmers (ATCC13076), and Pseudomonas aeruginosa (Schroeter) Migula (ATCC47085) were cultured in Mueller–Hinton (MH) broth containing 1.5% agar. Each bacterium was mixed in 0.5% soft agar which was then poured on MH agar plates.

Eggs (no wax extraction or wax extracted) were placed directly on the agar surfaces overlaid with bacterial culture. The organic extract (10 mg dissolved in 50 μl chloroform/methanol) or the aqueous extract (2 mg dissolved in 50 μl sterilized water) was added to filter paper discs (6 mm), air dried for 15 min, and subsequently placed on the bacterial culture overlay plates. Plates were incubated for 48 h at 37°C, and the resultant inhibition zones were measured with calipers to the nearest 0.1 mm. Data were analyzed by analysis of variance using Statistica 6.0 software (Statsoft, Tulsa, OK).

Wax extract quantity

No significant differences were observed in the total amount of wax extracted from the eggs, as well as the amounts within the organic and the aqueous phases of the extracts of the different tick species (F = 7.19, df = 4, P = 0.41). Amounts of wax extracted are listed in Table 1.

Table 1 Mean ± SEM amounts (mg/g) of wax extracts from the eggs of selected ixodid ticks.

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Antibacterial activity of eggs

Eggs from which the waxes had been extracted showed no inhibitory effect against the bacteria tested in our study, regardless of the tick species. Eggs that had not undergone wax extraction, however, inhibited bacterial growth in these in vitro assays. Haemaphysalis longicornis (parthenogenic and bisexual populations) and H. doenitzi eggs inhibited the growth of gram-positive bacteria, but not gram-negative bacteria (Table 2). On the other hand, eggs from D. silvarum and Hy. asiaticum inhibited the growth of gram-negative bacteria, but not gram-positive bacteria (Table 3).

Table 2 Mean ± SEM diameter of inhibition zone (mm) of the unmanipulated eggs and egg wax of selected ixodid ticks against gram-positive bacteria.

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Table 3 Mean ± SEM diameter of inhibition zone (mm) of the unmanipulated eggs and egg wax of selected ixodid ticks against gram-negative bacteria.

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Antibacterial activity of wax extracts

Both the organic and aqueous extracts of the waxes from D. silvarum and Hy. asiaticum eggs inhibited the growth of gram-positive bacteria but not gram-negative bacteria (Table 2). And, the organic and aqueous extracts from H. longicornis (parthenogenic and bisexual populations) and H. doenitzi eggs inhibited the growth of gram-negative bacteria, but not gram-positive bacteria (Table 3).