Ueno Takayuki, Kawasaki Kiyoshi, Kubo Takeo
Department of Biological Sciences, Graduate School of Sciences, The University of Tokyo; Faculty of Pharmaceutical Sciences, Doshisha Women's College;
Faculty of Pharmaceutical Sciences, Doshisha Women's College.
J Vis Exp. 2016 Sep 6(115):54240. doi: 10.3791/54240.
Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related division of labor). Young workers are engaged in nursing the brood (nurse bees), while older workers are engaged in foraging for nectar and pollen (foragers). The physiology of the workers changes in association with this role shift. For example, the main function of the hypopharyngeal glands (HPGs) changes from the secretion of major royal jelly proteins (MRJPs) to the secretion of carbohydrate-metabolizing enzymes. Because worker tasks change as the workers age in typical colonies, it is difficult to discriminate the physiological changes that occur with aging from those that occur with the role shift. To study the physiological changes in worker tissues, including the HPGs, in association with the role shift, it would be useful to manipulate the honeybee colony population by preparing single-cohort colonies in which workers of almost the same age perform different tasks. Here we describe a detailed protocol for preparing single-cohort colonies for this analysis. Six to eight days after single-cohort colony preparation, precocious foragers that perform foraging tasks earlier than usual appear in the colony. Representative results indicated role-associated changes in HPG gene expression, suggesting role-associated HPG function. In addition to manipulating the colony population, analysis of the endocrine system is important for investigating role-associated physiology. Here, we also describe a detailed protocol for treating workers with 20-hydroxyecdysone (20E), an active form of ecdysone, and methoprene, a juvenile hormone analogue. The survival rate of treated bees was sufficient to examine gene expression in the HPGs. Gene expression changes were observed in response to 20E- and/or methoprene-treatment, suggesting that hormone treatments induce physiological changes of the HPGs. The protocol for hormone treatment described here is appropriate for examining hormonal effects on worker physiology.
蜜蜂工蜂承担着与维持蜂群活动相关的各种任务。工蜂的任务会根据其年龄而变化（与年龄相关的劳动分工）。年轻工蜂负责照料幼虫（保育蜂），而年长工蜂则负责采集花蜜和花粉（采蜜蜂）。工蜂的生理机能会随着这种角色转变而发生变化。例如，下咽腺（HPGs）的主要功能从分泌主要蜂王浆蛋白（MRJPs）转变为分泌碳水化合物代谢酶。由于在典型蜂群中工蜂的任务会随着年龄增长而改变，因此很难区分衰老过程中发生的生理变化与角色转变过程中发生的生理变化。为了研究包括下咽腺在内的工蜂组织与角色转变相关的生理变化，通过制备单一群落蜂群（其中几乎同龄的工蜂执行不同任务）来操控蜜蜂群体数量会很有帮助。在此，我们描述了用于此分析的制备单一群落蜂群的详细方案。在制备单一群落蜂群六至八天后，蜂群中会出现比平常更早执行采集任务的早熟采蜜蜂。代表性结果表明下咽腺基因表达存在与角色相关的变化，提示了与角色相关的下咽腺功能。除了操控蜂群数量外，分析内分泌系统对于研究与角色相关的生理学也很重要。在此，我们还描述了用蜕皮激素的活性形式20 - 羟基蜕皮酮（20E）和保幼激素类似物烯虫酯处理工蜂的详细方案。经处理蜜蜂的存活率足以用于检测下咽腺中的基因表达。观察到了对20E和/或烯虫酯处理有响应的基因表达变化，表明激素处理会诱导下咽腺的生理变化。此处描述的激素处理方案适用于检测激素对工蜂生理的影响。
