蜜蜂幼虫末龄体重与其后期变态发育的关系研究

It is an important problem of developmental biology how insect can realize the accurate control of its size. Our preliminary investigations demonstrated that caste determination in honey bee is regulated by critical weight. The last-instar larvae of 160-180 mg and 320-340 mg will be development respectively for the worker bee and the queen bee under the condition of artificial feeding. However, the relationship between female larvae weight and its later metamorphosis is unknown. In the present study, honeybee larvae (Apis mellifera L.) with different body weights (ranges from 160 mg to 340 mg) were used to mimic the last-instar larvae of the honey. The technology of histological section, chromatographic analysis and high-throughput sequencing were performed to analysis the relationship between the weight of final instar larvae and its effects on the developmental periods, fecundity, behavioral expression. Meanwhile, the influence rule and mechanism of larvae body weight on the secretion of juvenile hormone and 20-hydroxyecdysone were studied. Finally, the DNA methylation levels and patterns of the larva in different stages were examined. The molecular mechanism regulating development of larvae with different final weight will be elucidated preliminarily. The relevant results will not only helpful for understanding the relationship between the weight of the last-instar larva and its later metamorphosis in honey bees, but also provides an important theoretical support for the mechanism study of the caste determination in honey bees.

昆虫如何感知并精确控制其个体大小是重要的发育生物学问题。申请人前期研究发现，蜜蜂级型分化过程存在末龄幼虫临界体重调控机制，人工饲养条件下，160-180mg和320-340mg的末龄幼虫会分别发育为工蜂型和蜂王型蜜蜂，但末龄幼虫体重与其后期变态发育的关系如何？尚不清楚。本项目以意大利蜜蜂为研究对象，对体重在160-340mg的幼虫进行饥饿处理，模拟不同体重末龄幼虫，利用组织切片、色谱分析、高通量测序等技术，研究末龄幼虫体重与其后期变态发育过程中发育历期、形态特征、繁殖力、行为表现等指标的关系及变化规律；研究幼虫体重对保幼激素和蜕皮激素滴度的影响及规律，明确其生理调控机制；分析幼虫体重与其后期发育过程中DNA甲基化水平及模式差异并阐明其分子调控机理。所得结果不仅可以揭示雌性蜜蜂末龄幼虫体重与其后期变态发育过程的关系及调控机制，也对完善蜜蜂级型分化研究理论提供重要科学借鉴。

动物个体大小的调控机制研究是发育生物学领域研究的重大热点科学问题，蜜蜂幼虫感知并实现对其个体和器官大小的精确控制涉及极其复杂的生物学调控。本项目以意大利蜜蜂为研究对象，探索了雌性蜜蜂末龄幼虫体重与其后期变态发育的关系，研究了不同体重条件下幼虫的发育历期、成蜂体重、形态及行为特征变化情况，明确了幼虫体重与其生长发育、形态特征、繁殖力、行为表现等生理生态特征的动态关系及规律；研究了不同体重幼虫发育后期JH和20E滴度的变化规律，探明其生理调控机制；研究蜂王型、中间型和工蜂型体重幼虫后期变态发育过程中转录组和DNA甲基化水平变化及基因表达差异，从基因组水平上探讨调控二态性体重幼虫后期变态发育过程调控的分子作用机制。研究揭示了蜜蜂末龄幼虫体重与蜜蜂成蜂个体大小及发育调控存在密切联系，研究证实：对工蜂幼虫而言，在满足变态发育营养需求情况下，营养限制起生长调节作用，减缓其生长发育速度，致使其在特定时期发生不可逆性生理伤害；空间限制起哺育终止作用，决定其生长发育期末个体大小。两者共同作用，最终导致工蜂幼虫体重及后期变态过程与蜂王产生差异——即级型分化现象。进一步的，在发育的不同时期，两型蜂幼虫DNA甲基化和转录组调控模式相似，但两型蜂幼虫，特别是工蜂幼虫在不同发育时期DNA甲基化水平和转录组基因表达水平发生显著差异性变化可能导致了蜜蜂级型分化现象的产生。