Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

Summary

Here we describe our detailed protocol for the preparation of single-cohort honeybee colonies – a useful tool for analyzing the role-associated worker physiology. We also describe detailed protocols for treating workers with juvenile hormone and ecdysone to evaluate the involvement of these hormones in the regulation of worker behavior and/or physiology.

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

The European honeybee, Apis mellifera, is a eusocial insect with a highly organized society¹. Worker honeybees (labor caste) are engaged in various tasks to maintain colony activity, and these tasks change according to the worker honeybee's age after eclosion, which is referred to as age-related division of labor^2-4. Young workers (<13 days old) take care of the brood in the hive by secreting royal jelly (nurse bees), while older workers (>15 days old) collect nectar and pollen outside of the hive (foragers)^2-4. The physiology of the workers changes in association with this role shift. For example, the function of ....

Protocol

1. Preparation of Single-cohort Colonies

1. Prepare three honeybee colonies to create two single-cohort colonies and to obtain a sufficient number of newly emerged workers.
   1. Check that some pupae in the capped peripheral cells in the combs have brown eyes and a pigmented cuticle by opening the capped combs using tweezers. If these pupae exist in peripheral comb cells, most of pupae in the whole combs will emerge in approximately 1-3 days.
   2. Subsequently, collect the combs containing these pupae after all adherent adult bees have been removed with a brush, and mix combs from the three colonies to minimize variability among colonies.
   <....

Results

An overview of the protocol for preparing single-cohort colonies is illustrated in Figure 1A. Time-course of experiments from preparing single-cohort colonies to sample collection is shown in Figure 1B. Workers that satisfied the behavioral criteria for nursing behavior or foraging behavior were collected from single-cohort colonies, and HPG development was estimated in these workers. Table 1 shows the classification of HPG development in.......

Discussion

Preparation of single-cohort colonies

Here we described the protocol used in our previous study¹⁶ to prepare single-cohort colonies for analysis of HPG physiology associated with the shift in the role of worker bees. Nurse bees and precocious foragers that satisfied the criteria described in procedures 1.6-1.7 and Figure 2 were observed in the single-cohort colonies (Table 1). The photographs in Figure 2 would be useful in classific.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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