南洋臀纹粉蚧生物学特性及LAMP检测

Biological characters and loop-mediated isothermal amplification detection of Planococcus lilacinus(Hemiptera:Pseudococcidae)

【目的】明确南洋臀纹粉蚧Planococcus lilacinus生物学特性及实现对该虫的可视化检测。【方法】室内观察记录南洋臀纹粉蚧不同发育阶段的形态特征和历期及雌成虫的繁殖能力;以南洋臀纹粉蚧28S rDNA序列为靶标,设计其特异性的环介导等温扩增(loop-mediated isothermal amplification,LAMP)引物组用于建立相应的可视化检测方法,并进行特异性、灵敏度及稳定性验证。【结果】南洋臀纹粉蚧雌雄虫具有不同的世代生活史,卵初产时即可见虫体轮廓,并具1对暗红色单眼;1龄若虫行动活泼,雌雄难辨;2龄若虫体缘出现白色细蜡棒,雌雄体型出现分化;3龄若虫仅存在雌虫,形似雌成虫;蛹仅存在雄虫,分为预蛹期和蛹期;雌成虫体表覆盖厚实的白色蜡粉,体缘18对粗蜡棒突出明显;雄成虫有一对透明发达的前翅,腹部末端具1对白色长蜡丝。该虫卵历期(1.37±0.26)h,雌成虫历期(32.67±4.82)d,各龄若虫、蛹和雄成虫历期均不超过(7.65±0.82)d;两性生殖的单雌产卵量和卵孵化率分别为(531.92±69.98)粒和96.02%±1.35%,孤雌生殖的单雌产卵量和卵孵化率分别为(403.50±71.11)粒和88.41%±3.03%。建立的LAMP可视化检测方法能有效扩增南洋臀纹粉蚧DNA,最佳反应温度和时间及最低检测浓度分别为66℃、55 min和100 fg/μL,反应产物呈绿色阳性,检出率达91.67%~100.00%,但无法扩增其他7种近缘种属粉蚧DNA,反应产物呈橙黄色阴性。【结论】南洋臀纹粉蚧雌雄二型,生长发育快,繁殖力强,急需作好其检测与监测工作;该虫的LAMP可视化检测方法便捷高效、特异性强、灵敏度高且稳定性好,有望用于非专业鉴定人员对该虫的现场快速、准确识别。

【Aim】To define the biological characters of Planococcus lilacinus and achieve its visual detection.【Methods】The morphological characteristics and duration of different developmental stages and female adult fecundity of P.lilacinus were determined in the laboratory.Then based on the 28S rDNA sequence of P.liacinus,the specific primers of loop-mediated isothermal amplification(LAMP)were designed to establish the corresponding visual detection method,and the specificity,sensitivity and stability of LAMP detection were verified.【Results】The female and male P.lilacinus have different generational life histories.From the egg when it is laid,the outline of insect body and one pair of dark red ocelli just can be seen.The 1st instar nymph moves fast and it is difficult to identify its female and male.The 2nd instar nymph has thin white wax rods on the body margins with differentiated body morph between female and male.The 3rd instar nymph only belongs to female and has similar shape to female adult.The pupa only belongs to male and includes prepupal and pupal stages.The female adult is covered by thick white wax powders,with 18 pairs of visible wax rods on the body margins.The male adult has one pair of transparent and developed fore wings,and one pair of long white wax filament at the terminal part of the abdomen.The duration of egg and female adult lasted(1.37±0.26)h and(32.67±4.82)d,respectively,while that of each instar nymph,pupa and male adult all lasted no more than(7.65±0.82)d.Meanwhile,the number of eggs laid per female and the egg hatching rate in sexual reproduction were(531.92±69.98)grains and 96.02%±1.35%,respectively,while those in parthenogenetic reproduction were(403.50±71.11)grains and 88.41%±3.03%,respectively.Furthermore,the established LAMP visual detection method could effectively amplify the DNA of P.lilacinus,it had the optimal reaction temperature of 66℃,optimal reaction time of 55 min,detection limit of at least 100 fg/μL and detection rate of 91.67%-100.00%with the positive green reaction products.But the LAMP detection could not amplify the DNA of the other seven mealybug species with the negative saffron yellow reaction products.【Conclusion】There is sexual dimorphism in P.lilacinus life cycle with fast growth and development and strong fecundity,therefore it is necessary to be detected and monitored.The LAMP visual detection method is rapid,specific,sensitive and reliable,it shows an excellent potential in rapid and accurate identification of this mealybug for non-professional identifier on-site.