森林革蜱种群生态及希伯来花蜱卵蜡层抑菌分子研究
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摘要
本研究利用野外可控及模拟实验调查了河北省自然条件下森林革蜱Dermacentor silvarum生活史各发育阶段的生物学特性。在蜱类分布的自然生境中建立可控样方,自然光照和气候条件下,森林革蜱D. silvarum幼蜱、若蜱和成蜱在兔体吸血,非寄生阶段在可控样方内自由生活,记录其各发育阶段生活史参数并观察找寻宿主行为及季节发生情况。结果表明雌蜱具有行为滞育,且其产卵时间及卵的孵化都具有一致性,从而使自然条件森林革蜱D. silvarum主要活跃在2月下旬至9月上旬。森林革蜱D. silvarum雌蜱可在3月、4月和5月饱血,不同月份饱血的雌蜱其产卵前期和卵的孵化期差异极大,进而使其生活史周期也存在较大差异。自然条件下幼蜱(18天)和若蜱(29天)的发育历期与实验室条件下无明显差异。雌蜱饱血体重与产卵量相关(r = 0.76, p < 0.001),其在3月、4月和5月的生殖效率指数分别为5.7、7.1和6.2。自然条件下,森林革蜱D. silvarum雌蜱产卵方式与实验室条件下有很大不同。
在此基础上,进一步调查了自然条件下寄生期及非寄生期森林革蜱D. silvarum的季节发生数量及动态。结果表明,森林革蜱D. silvarum主要在灌丛滋生,一年仅能完成一个世代,其生活史各阶段季节发生具有连续性,仅幼蜱和若蜱的活动存在部分交叉。成蜱主要活跃在2月下旬到5月下旬,高峰出现在4月中旬;幼蜱在6月上旬至9月上旬活动,高峰出现在7月中旬;若蜱主要分布在6月下旬至9月下旬,高峰在8月中旬。森林革蜱D. silvarum成蜱和若蜱主要侵袭羊体耳部,幼蜱不能在羊体寄生。10月后,在羊体发现部分越冬雄蜱寄生,但拖旗法未能捕获自由生活成蜱。
测定了希伯来花蜱Amblyomma hebraeum卵及卵蜡层提取物的抑菌活性及抑菌谱,结果表明,希伯来花蜱A. hebraeum卵能够抑制革兰氏阴性菌生长,卵蜡层有机相和水相提取物抑制革兰氏阳性菌的生长。有机相提取物中主要为胆固醇酯以及碳链长度为13到26的脂肪酸。脂类分馏和气相色谱/质谱结果进一步表明,有机相提取物中,不饱和脂肪酸C16:1和C18:2存在抑菌活性。水相提取物与有机相提取物的组成存在差别,且水相提取物具有极强的热稳定性、pH稳定性及蛋白酶稳定性。核磁共振分析测定了36种代谢物,结果表明,其均不具有抑菌活性,因此,水相提取物中发挥抑菌活性的物质还需进一步分离纯化。
In this study, we investigated the life cycle and biological characteristics of all developmental stages of Dermacentor silvarum under field conditions in Hebei province in North China. Larvae, nymphs and adults were fed on rabbits, and maintained under natural lighting and climate conditions. Ticks were released into a confined plot located in natural tick habitat, and we observed the host questing behavior and seasonal occurrence. Due to a behavioral diapause in females and the synchronization of oviposition and larval hatching, D. silvarum only active from late February to early September under field conditions. The duration of the life cycle of D. silvarum was highly variable due to different preovipositional and egg hatching periods for ticks engorging in March, April or May. The developmental periods of larvae (18 days) and nymphs (29 days) under field conditions were similar to those under laboratory conditions. Moreover, there was a significant correlation between engorged body weight of females and egg masses laid (r = 0.76, p < 0.001). The female reproductive efficiency index (REI) in March, April and May was 5.7, 7.1 and 6.2, respectively. Finally, we also observed different ovipositional patterns between females that engorged in the field and those that engorged under laboratory conditions.
Studies on seasonal abundance and activity of D. silvarum of larvae, nymphs and adults both on and off the host were carried out in northern China. Results of this study suggested that the ticks mainly reside in shrubs, and can only complete one generation per year with a sequential seasonal distribution, and little overlap between the activity of the larvae and nymphs. Adults were most prevalent from late February to late May with a peak in number in the middle of April; the larvae were found from early June to early September with a peak in the middle of July, while the nymphs were mainly distributed from late June to late September with peak in the middle of August. Adult and nymphal D. silvarum were found primarily on the ears of the sheep, but no larvae were found on sheep. Additionally, an overwintering male adults population was detected on sheep after October, but no free-living adults were found by dragging.
Antimicrobial activity and spectrum of both eggs of the tick Amblyomma hebraeum and egg wax extract was investigated in the current work, results showed that eggs can inhibit the growth of Gram-negative bacteria, and the egg wax extract including both the organic and aqueous phase can only function against Gram-positive bacteria. Lipid class and fatty acid composition of the organic extract of the egg wax was analysed by High-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS), cholesterol esters was the most abundant lipid followed by fatty acids with chain lengths between C13 to C26. After lipid fractionation and GC/MS, free fatty acids, especially unsaturated fatty acids C16:1 and C18:2 were found to account for the antimicrobial activity in the organic extract of the egg wax. Antimicrobial substances contained in the aqueous extract of the egg wax were different from that in the organic extract, with extremely heat resistant, pH stability and proteinase stability. Although 36 metabolites were identified in the aqueous extract by Nuclear Magnetic Resonance (NMR) analysis, they were proved to contribute no effect on the antimicrobial activity in the aquoues extract. This suggests that the functional substances in the aqueous extract renains unclear, and the antibacterial activity changes following wax extraction remains unexplainable.
在此基础上,进一步调查了自然条件下寄生期及非寄生期森林革蜱D. silvarum的季节发生数量及动态。结果表明,森林革蜱D. silvarum主要在灌丛滋生,一年仅能完成一个世代,其生活史各阶段季节发生具有连续性,仅幼蜱和若蜱的活动存在部分交叉。成蜱主要活跃在2月下旬到5月下旬,高峰出现在4月中旬;幼蜱在6月上旬至9月上旬活动,高峰出现在7月中旬;若蜱主要分布在6月下旬至9月下旬,高峰在8月中旬。森林革蜱D. silvarum成蜱和若蜱主要侵袭羊体耳部,幼蜱不能在羊体寄生。10月后,在羊体发现部分越冬雄蜱寄生,但拖旗法未能捕获自由生活成蜱。
测定了希伯来花蜱Amblyomma hebraeum卵及卵蜡层提取物的抑菌活性及抑菌谱,结果表明,希伯来花蜱A. hebraeum卵能够抑制革兰氏阴性菌生长,卵蜡层有机相和水相提取物抑制革兰氏阳性菌的生长。有机相提取物中主要为胆固醇酯以及碳链长度为13到26的脂肪酸。脂类分馏和气相色谱/质谱结果进一步表明,有机相提取物中,不饱和脂肪酸C16:1和C18:2存在抑菌活性。水相提取物与有机相提取物的组成存在差别,且水相提取物具有极强的热稳定性、pH稳定性及蛋白酶稳定性。核磁共振分析测定了36种代谢物,结果表明,其均不具有抑菌活性,因此,水相提取物中发挥抑菌活性的物质还需进一步分离纯化。
In this study, we investigated the life cycle and biological characteristics of all developmental stages of Dermacentor silvarum under field conditions in Hebei province in North China. Larvae, nymphs and adults were fed on rabbits, and maintained under natural lighting and climate conditions. Ticks were released into a confined plot located in natural tick habitat, and we observed the host questing behavior and seasonal occurrence. Due to a behavioral diapause in females and the synchronization of oviposition and larval hatching, D. silvarum only active from late February to early September under field conditions. The duration of the life cycle of D. silvarum was highly variable due to different preovipositional and egg hatching periods for ticks engorging in March, April or May. The developmental periods of larvae (18 days) and nymphs (29 days) under field conditions were similar to those under laboratory conditions. Moreover, there was a significant correlation between engorged body weight of females and egg masses laid (r = 0.76, p < 0.001). The female reproductive efficiency index (REI) in March, April and May was 5.7, 7.1 and 6.2, respectively. Finally, we also observed different ovipositional patterns between females that engorged in the field and those that engorged under laboratory conditions.
Studies on seasonal abundance and activity of D. silvarum of larvae, nymphs and adults both on and off the host were carried out in northern China. Results of this study suggested that the ticks mainly reside in shrubs, and can only complete one generation per year with a sequential seasonal distribution, and little overlap between the activity of the larvae and nymphs. Adults were most prevalent from late February to late May with a peak in number in the middle of April; the larvae were found from early June to early September with a peak in the middle of July, while the nymphs were mainly distributed from late June to late September with peak in the middle of August. Adult and nymphal D. silvarum were found primarily on the ears of the sheep, but no larvae were found on sheep. Additionally, an overwintering male adults population was detected on sheep after October, but no free-living adults were found by dragging.
Antimicrobial activity and spectrum of both eggs of the tick Amblyomma hebraeum and egg wax extract was investigated in the current work, results showed that eggs can inhibit the growth of Gram-negative bacteria, and the egg wax extract including both the organic and aqueous phase can only function against Gram-positive bacteria. Lipid class and fatty acid composition of the organic extract of the egg wax was analysed by High-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS), cholesterol esters was the most abundant lipid followed by fatty acids with chain lengths between C13 to C26. After lipid fractionation and GC/MS, free fatty acids, especially unsaturated fatty acids C16:1 and C18:2 were found to account for the antimicrobial activity in the organic extract of the egg wax. Antimicrobial substances contained in the aqueous extract of the egg wax were different from that in the organic extract, with extremely heat resistant, pH stability and proteinase stability. Although 36 metabolites were identified in the aqueous extract by Nuclear Magnetic Resonance (NMR) analysis, they were proved to contribute no effect on the antimicrobial activity in the aquoues extract. This suggests that the functional substances in the aqueous extract renains unclear, and the antibacterial activity changes following wax extraction remains unexplainable.
引文
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