单队列殖民地和工蜂的激素治疗的准备来分析生理学与关联作用和/或内分泌系统
Summary
在这里，我们描述详细的协议单队列蜂群的准备 – 分析相关角色工人生理的有用工具。我们还描述了用于治疗工人幼激素和蜕皮激素评估这些激素在工作者行为和/或生理调控的参与详细协议。
Abstract
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.
Introduction
欧洲蜜蜂， 蜜蜂 ，是一种完全社会性昆虫具有高度组织性的社会1。工蜂（劳动等级）从事各种工作，以保持集落的活性，并且这些任务根据羽化后工蜂的年龄，这被称为劳动2-4的年龄相关的分割发生变化。青年工人（<13日龄）照顾在蜂巢的育雏通过分泌蜂王浆（蜂护士），而年纪较大的工人（> 15天）采集花蜜和花粉蜂巢（征粮）2-4之外。工人的生理与这个角色转变的关联变化。例如，下咽腺（HPGS）的功能，配对位于头外分泌腺，改变与护理的作用转变为觅食2,5关联。护士蜂HPGS主要合成王浆主蛋白，是蜂王浆主要成分。另一方面，觅食HPGS主要合成碳水化合物代谢酶，如α葡糖苷酶III，通过将蔗糖转化为葡萄糖和果糖处理花蜜到蜂蜜。我们以前的研究显示，MRJP2的表达，其编码的一个主要蜂王浆蛋白质和Hbg3，它的作用移6-9中编码α葡糖苷酶III，变更。
要确定是否在工人组织中的生理变化，包括HPGS，与角色的转变或与工人的年龄，这将是操纵蜜蜂殖民地的人口组成，如准备单队列有用菌落在几乎相同年龄的工人执行不同的任务10,11。 Robinson 等人。（1989）描述了用于建立单一的队列菌落10的方法。单队列殖民地最初包括女王和0-2日龄工人。建立殖民地几天后，ALM工人OST同龄承担不同的任务。有些工人进行护理任务，如在典型菌落，而其他工作人员执行任务觅食比往年提前，并因此被称为早熟觅食。护士蜜蜂觅食早熟之间的基因表达的比较将提供有关工作的组织12-16的相关角色的生理有用的信息。这里，我们描述了制备单队列菌落HPGS 16的角色和/或与年龄相关的生理分析的详细协议。我们还简要介绍了如何通过定量逆转录-聚合酶链反应（RT-PCR）检查MRJP2和Hbg3的基因的表达来评价HPG生理学。
除了工人生理学单队列菌落的分析，内分泌系统的检查是用于分析相关联的角色的工人生理学的调节机制重要。幼激素（JH），其硝酸钾WN在昆虫幼虫的'现状'的激素，加速从护理的作用，在工蜂11觅食的转变。此外，蜕皮激素，这是变态过程称为蜕皮激素，可能参与的作用移作为编码蘑菇体被表达，工人脑17-19的较高中心蜕皮激素信号传导分子的基因。因此，我们还描述了在我们以前的研究16用于与20E治疗工人的详细协议，它是蜕皮激素的活性形式，和烯虫酯，一JH类似物，为的内分泌系统的上HPG生理效应分析（表达MRJP2和Hbg3）。
Protocol
Representative Results
Discussion
单队列殖民地的制备
在这里，我们描述了我们先前的研究16用于为与工蜂的角色转变相关的生理HPG分析准备单队列殖民地的协议。在单队列菌落（ 表1）中观察到护士蜜蜂和早熟觅食出满足在程序1.6-1.7和图2中所描述的标准。在HPG分类为基准规定的图2中的照片是有用的。基于这两个工人行为和HPG发展的标准是有效的，用于定义典型…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific Research (B) and a Grant-in Aid for Scientific Research on Innovative Areas ‘Systems Molecular Ethology’ from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. T.U. was the recipient of a Grant-Aid from the Japan Society for the Promotion of Science for Young Scientists.
Materials
| UNIPOSCA | Mitsubishi pencil | PC-5M | Marker pen for the application of marks to bees |
| 20-hydroxyecdysone | Sigma Aldrich | H5142 | |
| Methoprene | Sigma Aldrich | 33375 | |
| Breeding case insect | IRIS OHYAMA | CP-SS | |
| Electromotion mixier | ISO | 23M-R25 | homogenization of tissue |
| TRIZol Reagent | Invitrogen | 15596-026 | the reagent for total RNA extraction |
| DNase I | Takara | 2270A | |
| PrimeScript RT reagent kit | Takara | RR037A | the reagent for reverse transcription |
| SYBR Premix ExTaq II | Takara | RR820A | the reagent for real-time PCR |
| LightCycle 1.2 Instrument | Roche | 12011468001 | the instrument for real-time PCR |
| LightCycle Capillaries (20μl) | Roche | 4929292001 | the material for real-time PCR |
References
- Wilson, E. O. . The insect societies. , (1971).
- Winston, M. L. . The biology of the honey bee. , (1987).
- Lindauer, M. Ein Beitrag zur Frage der Arbeitsteilung im Bienenstaat. Zeitschrift für vergleichende Physiologie. 34 (4), 299-345 (1952).
- Sakagami, S. Untersuchungen uber die Arbeitsteilung in einen Zwergvolk der Honigbienen. Beitrage zur Biologie des Bienenvolkes, Apis mellifera L. I. Jap J Zool. 11, 117-185 (1953).
- Kubo, T., et al. Change in the expression of hypopharyngeal-gland proteins of the worker honeybees (Apis mellifera L.) with age and/or role. J Biochem. 119 (2), 291-295 (1996).
- Ohashi, K., Natori, S., Kubo, T. Change in the mode of gene expression of the hypopharyngeal gland cells with an age-dependent role change of the worker honeybee Apis mellifera L. Eur J Biochem. 249 (3), 797-802 (1997).
- Ohashi, K., Natori, S., Kubo, T. Expression of amylase and glucose oxidase in the hypopharyngeal gland with an age-dependent role change of the worker honeybee (Apis mellifera L). Eur J Biochem. 265 (1), 127-133 (1999).
- Ohashi, K., Sawata, M., Takeuchi, H., Natori, S., Kubo, T. Molecular cloning of cDNA and analysis of expression of the gene for alpha-glucosidase from the hypopharyngeal gland of the honeybee Apis mellifera L. Biochem Biophys Res Commun. 221 (2), 380-385 (1996).
- Ueno, T., Nakaoka, T., Takeuchi, H., Kubo, T. Differential gene expression in the hypopharyngeal glands of worker honeybees (Apis mellifera L.) associated with an age-dependent role change. Zoolog Sci. 26 (8), 557-563 (2009).
- Robinson, G. E., Page, R. E., Strambi, C., Strambi, A. Hormonal and genetic control of behavioral integration in honey bee colonies. Science. 246 (4926), 109-112 (1989).
- Sullivan, J. P., Fahrbach, S. E., Robinson, G. E. Juvenile hormone paces behavioral development in the adult worker honey bee. Horm Behav. 37 (1), 1-14 (2000).
- Ben-Shahar, Y., Robichon, A., Sokolowski, M. B., Robinson, G. E. Influence of gene action across different time scales on behavior. Science. 296 (5568), 741-744 (2002).
- Lehman, H. K., et al. Division of labor in the honey bee (Apis mellifera): the role of tyramine beta-hydroxylase. J Exp Biol. 209 (Pt 14), 2774-2784 (2006).
- Mutti, N. S., Wang, Y., Kaftanoglu, O., Amdam, G. V. Honey bee PTEN–description, developmental knockdown, and tissue-specific expression of splice-variants correlated with alternative social phenotypes). PLoS One. 6 (7), e22195 (2011).
- Whitfield, C. W., Cziko, A. M., Robinson, G. E. Gene expression profiles in the brain predict behavior in individual honey bees. Science. 302 (5643), 296-299 (2003).
- Ueno, T., Takeuchi, H., Kawasaki, K., Kubo, T. Changes in the Gene Expression Profiles of the Hypopharyngeal Gland of Worker Honeybees in Association with Worker Behavior and Hormonal Factors. PLoS One. 10 (6), e0130206 (2015).
- Paul, R. K., Takeuchi, H., Matsuo, Y., Kubo, T. Gene expression of ecdysteroid-regulated gene E74 of the honeybee in ovary and brain. Insect Mol Biol. 14 (1), 9-15 (2005).
- Takeuchi, H., et al. Identification of a novel gene, Mblk-1, that encodes a putative transcription factor expressed preferentially in the large-type Kenyon cells of the honeybee brain. Insect Mol Biol. 10 (5), 487-494 (2001).
- Takeuchi, H., Paul, R. K., Matsuzaka, E., Kubo, T. EcR-A expression in the brain and ovary of the honeybee (Apis mellifera L). Zoolog Sci. 24 (6), 596-603 (2007).
- Yamane, T., Miyatake, T. Reduced female mating receptivity and activation of oviposition in two Callosobruchus species due to injection of biogenic amines. Journal of Insect Physiology. 56 (3), 271-276 (2010).
- Ben-Shahar, Y., Leung, H. T., Pak, W. L., Sokolowski, M. B., Robinson, G. E. cGMP-dependent changes in phototaxis: a possible role for the foraging gene in honey bee division of labor. J Exp Biol. 206 (Pt 14), 2507-2515 (2003).
- Danforth, B. N., Ji, S. Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects. Mol Biol Evol. 15 (3), 225-235 (1998).
- Pandey, A., Bloch, G. Juvenile hormone and ecdysteroids as major regulators of brain and behavior in bees. Current Opinion in Insect Science. 12, 26-37 (2015).
