黑化粘虫防御反应及其神经内分泌调节机制

The oriental armyworm, Mythimna separata (Lepidoptera: Noctuidae), a typical long-distance migratory insect with density-dependent phase polyphenism, is a major pest of grain in China and other Asian countries, causing huge crop production and economic losses nationwide annually. Based on a field phenomenon that all the larval body colour among the infested M. separata population have changed to different degree of black, and most of the melanic larvae natural parasitism and pathogen infection rate have decreassed significantly, as well as were not sensitive to many conventional biological pesticides, we hypothesized that M. separata may exhibits density-dependent defense response or prophylaxis (DDP). However, the immune mechanism and neuroendocrine rugulation underpring DDP and its ecological consequences in the species remain unknown. In this project, on the basis of previous finding that a density-dependent melanization and the corresponding defense capacity have been experimentally tested in this species through a establishement of parasitoid (Exorista civilis) and pathogen (Metarrhizium anisopliae) infection model in the laboratory, most of the larval immunological variables including humoral and cellular immunity index, such as phenoloxidase activity, antimicrobial activity, hemocytes types and number and encapsulation in different ganization in response to larval rearing density and after exposure to the parasitoid and pathogen stress will be further determined systematically. This performance could be benefit to unveil the triple organic connections among density-dependent melanization, defense capacity and immune mechanism. Moreover, to disclose the neuroendocrine hormonal regulation of the larval DDP in this species, larval defense capacity and immune response function will be identified after exogenous injection of neurohormones (Octopamine, dopamine and 5-hydroxytrypamine) and endocrine hormones (JH and MH), combined with the test of a variety of neuroendocrine hormones titer change during parasitoid and pathogen infection. Finally, survived larval feeding and emergenced adults flight and reproduction performance after exposure to the parasitoid and pathogen will be comprehensively tested so as to identify the post-immune consequence related to migration behaviour shifting in the species. The project will be helpful to illustrate DDP mechanism, its hormonal regulation and eclogical consequence, and this will provide theoretical and experimental support for unveiling M. separata outbreak mechanism in the field and guiding biological control.

粘虫是典型的密度依赖性变型重大农业迁飞害虫，田间发现所有成灾种群均发生了不同程度的黑化且天敌寄生率和病原物侵染率明显下降并对生物制剂不敏感，推测其具有密度依赖性防御反应，但这种防御机理、调控机制及生态意义均不清楚。项目在前期成功建立不同密度下粘虫天敌寄生和病原物侵染平台并发现其密度依赖性黑化与防御特性的基础上，进一步测定不同组织免疫指标（酚氧化酶、抗菌活力、血细胞种类、数量及包囊作用）变化，探明粘虫密度依赖性黑化-防御能力-免疫机理三者间有机联系。同时，通过外源注射神经激素（章鱼胺、多巴胺和5-羟色胺）和内分泌激素（保幼激素和蜕皮激素）明确其对幼虫防御能力和免疫指标的影响，并结合免疫过程中各种激素含量变化，揭示其神经内分泌调节机制。最后，综合测试免疫后幸存幼虫取食、成虫飞行与生殖能力，鉴定其行为变化。为阐明粘虫密度依赖性防御机理、调节机制和免疫效应以及解析成灾机制和指导生物防治提供依据。

粘虫是一种典型的远距离迁飞危害的重大农业害虫，为阐明其免疫致灾机制，项目在研究明确粘虫具有明显的密度依赖性黑化基础上，阐明了不同密度下幼虫抵抗伞群追寄蝇寄生及绿僵菌侵染能力的差异，揭示了粘虫存在明显的密度依赖性防御反应特征。进一步系统阐明了黑化粘虫（群居型）与淡色粘虫（散居型）受绿僵菌侵染后防御反应的体液和细胞免疫指标的差异，两型粘虫在血淋巴蛋白含量、PO活性、溶菌酶活性、抗菌活力以及血细胞数量和种类均有显著的差异，总体上黑化粘虫免疫免疫系统活跃，免疫应答具有更强优势。同时，研究明确了黑化粘虫和淡色幼虫免疫反应的神经和内分泌调节差异。黑化粘虫DA含量高，而5-HT含量相异。绿僵菌侵染后黑化粘虫DA和5-HT含量总体上呈升高趋势，而淡色粘虫DA含量也呈升高趋势，但5-HT呈先升高后下降趋势。进一步对黑化粘虫注射神经递质DA和5-HT后发现，黑化粘虫各免疫因子对神经递质有不同的时间响应动态。注射神经递质DA、5-HT后，黑化粘虫PO活性、抗菌活力在整体水平上均显著高于对照组，而蛋白含量和溶菌酶活力在注射后虽然也有上升，但总体上较对照组相比并无显著性差异。注射神经递质对黑化粘虫防御能力也有一定的影响，注射DA后显著提高幼虫存活率，而注射5-HT幼虫存活率变化不显著。其原因是注射DA后体液免疫指标增加更加明显。黑化粘虫受球孢白僵菌侵染后抑制了幼虫JH积累，但提高了MH含量。注射JHA后，黑化粘虫侵染后校正死亡率上升，化蛹率和羽化率均呈下降趋势。原因主要是JHA显著降低了幼虫体内的细胞结节数量、蛋白含量、溶菌酶活性、酚氧化酶活性和抗菌肽含量等免疫指标。注射20E后，黑化粘虫幼虫校正死亡率显著低于对照，免疫指标均在侵染后8~48h显著高于对照。这说明JH不利于黑化粘虫幼虫对球孢白僵菌的免疫反应，而MH则有利于提高其免疫能力。粘虫幼虫防御反应后幸存个体飞行和生殖均有显著变化，黑化粘虫免疫后幸存个体飞行能力较淡色粘虫显著增强，飞行时间和飞行距离均显著高于对照，但生殖能力和寿命没有显著变化。