蝗虫微孢子引起飞蝗表皮白化的生化分子机制

Paranosema locustae is an obligate parasite of Orthoptera, which has been researched and applied widely in controlling locusts and grasshoppers, the significant impact of Paranosema locustae to Locusta migratoria's morphology and phase has been proved, but the influence of insect pathogenic microsporidia on the melanin formation of the body wall of Locusta migratoria and biochemical and molecular mechanisms have not been documented so far. With a CCD imaging system and ImageJ image processing system, high performance liquid chromatography (HPLC), immunohistochemistry, spectrophotometry and SYBR Green fluorescence quantitative PCR, the biochemical and molecular mechanisms about Paranosema locustae interupting the cuticular albino of Locusta migratoria will be studied here. The gray of pronotum and sterna blackspot of the locusts infected with P. locustae, and the number of albino individuals in the infected locust population at different stage and infection must be observed and pictured firstly; further researches on the influences of P. locustae on different infection of locust’s juvenile hormone (JH) and [His7]-corazonin level in hemolymph are carried out; and then the interference of P. locustae to the activity of tyrosine hydroxylase, tyrosine hydroxylase and phenol oxidase, synthesis of indole 5-6 quinone, dopamine and eumelanin are studied; finally, the disruption of the P. locustae on the expression of henna and pale gene relate to the insect cuticular melanization,and then effects of the pathogen on microRNA miR-133 regulating transcription and translation of henna and pale gene are researched. The research results will be important to understand a new mechanism controlling locusts and grasshoppers with the pathogen, looking for new target and improving the level of locust management by the Microsporidia.

蝗虫微孢子是广泛用于防治蝗虫的专性病原物，该病原可显著干扰飞蝗型变过程，但其对与飞蝗型变密切相关的体色的影响及机制未见报道。本项目拟采用CCD成像和ImageJ图像处理、HPLC、免疫组织化学和SYBR Green qPCR等方法和技术，研究蝗虫微孢子对群居型飞蝗表皮的白化作用及其生化分子机制。首先研究感染了不同浓度蝗虫微孢子的不同生育期飞蝗的前胸背板灰度和腹部黑色斑的变化；进一步研究不同感病程度飞蝗血淋巴及神经节中保幼激素和黑化诱导激素含量差异；同时研究微孢子对飞蝗体壁或脑神经节中酪氨酸羟化酶、多巴脱羧酶和酚氧化酶活性及多巴胺、5,6-二羟基吲哚醌和真黑色素含量的影响；重点明确微孢子对调控飞蝗黑色素合成的henna和pale基因表达的抑制作用，以及对调控这些基因转录和翻译的miR-133表达的影响。研究结果对揭示蝗虫微孢子控制蝗害的新机制、寻找防治蝗虫新靶标和提升生物防治水平有重要作用。

蝗虫是世界上最重要的害虫之一，蝗虫微孢子虫是第一个且应用面积最大的用于防治蝗虫的生物制剂，明确其对蝗虫体色的影响及其机制，对于促进其防治效果有重要意义。项目利用CCD成像系统和ImageJ软件观察和分析了蝗虫微孢子对黑色群居型飞蝗的白化作用；采用生化分析和SYBR Green qRT-PCR等技术分别研究了蝗虫微孢子对调控黑色群居型飞蝗黑色素合成的生化因子及基因表达的影响，结果发现感染蝗虫微孢子虫后群居型飞蝗浅色型个体比例显著升高，飞蝗腹侧面色斑高度与腹部总高度比值明显降低，腹部体壁总黑色素含量降低20.46%；而腹部和胸部碱溶性黑色素降幅分别达75.98%和19.11,飞蝗胸部和腹部真黑色素含量分别降低75.85%和63.05%。感病蝗虫血淋巴中多巴胺和血清素含量下降明显。蝗虫微孢子病对主要中枢神经递质牛磺酸、酪胺酸、瓜氨酸和精氨酸合成的负面影响明显，这些神经递质对蝗虫的生长发育有重要促进作用。进一步研究发现染病飞蝗体壁组织中酪氨酸酶和酚氧化酶活力出现了明显的下降，过氧化酶体通路和P450解毒通路中的两个基因LOCMI09257和LOCMI16988分别发生了上调和下调，导致过氧化物减少和P450通路解毒能力明显下降，酪氨酸和苯丙氨酸代谢途径与病原物的侵染和体色变化有关，病原物通过影响苯丙氨酸代谢途径进而调控酪氨酸代谢途径从而引起飞蝗体色变化。深入研究发现，蝗虫微孢子虫侵染后的飞蝗，其体色的变化与飞蝗miRNAs相关，对健康和感病的黑色及浅色型飞蝗前胸背板的miRNAs高通量测序及荧光定量分析发现了多个差异表达的miRNAs，明确了多个新的miRNAs（如Novel_27/_41）与感病飞蝗色型分化相关。结果为深入研究飞蝗色素合成的分子机制奠定了基础。