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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 the hypopharyngeal glands (HPGs), paired exocrine glands located in the head, changes in association with the role shift from nursing to foraging^2,5. Nurse bee HPGs mainly synthesize major royal jelly proteins, which are major components of bee milk. On the other hand, forager HPGs mainly synthesize carbohydrate-metabolizing enzymes, such as α-glucosidase III, to process nectar into honey by converting sucrose into glucose and fructose. Our previous studies revealed that the expression of mrjp2, which encodes a major royal jelly protein, and Hbg3, which encodes α-glucosidase III, changes during the role shift^6-9.

To determine whether the physiological changes in the worker tissues, including the HPGs, is associated with the role shift or with the age of the workers, it would be useful to manipulate the population composition of a honeybee colony, such as to prepare single-cohort colonies in which workers of almost the same age perform different tasks^10,11. Robinson et al. (1989) described a method for establishing a single-cohort colony¹⁰. Single-cohort colonies initially comprise a queen and 0-2 day old workers. Several days after establishing the colonies, workers of almost the same age assume different tasks. Some workers perform nursing tasks as in typical colonies, whereas other workers perform foraging tasks earlier than usual and are thus called precocious foragers. Gene expression comparisons between nurse bees and precocious foragers would provide useful information about the role-associated physiology of worker tissues^12-16. Here, we describe a detailed protocol for preparing single-cohort colonies for analysis of the role- and/or age-associated physiology of HPGs¹⁶. We also briefly describe how to examine the gene expression of mrjp2 and Hbg3 by quantitative reverse transcription-polymerase chain reaction (RT-PCR) to evaluate HPG physiology.

In addition to the analysis of worker physiology in single-cohort colonies, examination of the endocrine system is important for analyzing the regulatory mechanisms of role-associated worker physiology. Juvenile hormone (JH), which is known as the 'status quo' hormone in insect larvae, accelerates the shift in the role from nursing to foraging in worker honeybees¹¹. Furthermore, ecdysone, which is known as the molting hormone during metamorphosis, might be involved in the role shift as genes encoding ecdysone signaling molecules are expressed in the mushroom bodies, a higher center of the worker brain^17-19. Therefore, we also describe the detailed protocol used in our previous study¹⁶ to treat workers with 20E, which is an active form of ecdysone, and methoprene, a JH analogue, for analysis of the effect of the endocrine system on HPG physiology (expression of mrjp2 and Hbg3).

Protocol

1. Preparation of Single-cohort Colonies Prepare three honeybee colonies to create two single-cohort colonies and to obtain a sufficient number of newly emerged workers. 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. 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. Place the collected combs in an empty hive box and incubate at 33 °C under humid conditions (>60% relative humidity). Collect approximately 6,000 newly emerged workers over 3 days (3,000 workers per colony) from the collected combs. Determine the quantity of workers based on the weight of five newly emerged workers randomly collected from unmanipulated colonies. Weigh five newly emerged workers randomly collected from unmanipulated colonies using weighing machines. Note: The weight of five newly emerged workers is generally 500-600 mg. Estimate the quantity of collected workers by dividing the weight of the collected workers by the average weight of five newly emerged workers. Apply paint marks to the thoraces of approximately 1,800 workers (900 workers per colony) using water-based poster paint markers. Note: Washable marker which is used to apply the paint is listed in the Table of Materials. Introduce a queen and approximately 3,000 of the 0-2 day old workers to a new hive box that contains two combs, one comb containing honey and pollen as preserved foods and another empty comb for egg-laying by the queen. Collect the comb containing honey and pollen from another unmanipulated colony after all adherent bees are removed. Note: The use of nuclear hive box (small sized hive box) is recommended. Collect nurse bees and precocious foragers with paint marks 6 to 8 days after creating single-cohort colonies. To collect nurse bees, pick up workers that put their heads into comb cells to take care of the brood. Use tweezers to collect the nurse bees from combs that contain many brood and adult workers. Note: If HPGs of collected workers are developed when dissected as described in step 1.7, these workers are defined as nurse bees. Holding the thoraces by tweezers is recommended to pick up workers from the combs. To collect foragers, use an insect net to capture workers that return to their hives with pollen loads on their hind legs. Note: If HPGs of captured workers appear shrunken when dissected as described in step 1.7, those workers are defined as foragers. Classify the HPG into three groups, 'Developed', 'Intermediate', and 'Shrunken' by the dissection under a stereomicroscope. Anesthetize 10-15 bees in an insect cage in a refrigerator for 10-15 min until bees cannot fly or walk. Then, move bees onto ice and complete the anesthesia on ice for 5 min. Note: It takes about total 15-20 min to achieve anesthesia. If the anesthesia time needs to be shortened, the number of bees in a cage should be reduced to 1-3 bees per cage. Dissect the head from the body with scissors and tweezers. Fix the head on dental wax placed on a Petri dish with pins. Insert two pins into the bases of the antennae to fix the head. Soak fixed heads in insect saline (130 mM NaCl, 5 mM KCl, 1 mM CaCl2). Remove the anterior aspect of the cuticle of the head using fine tweezers and a surgical knife under a binocular microscope. Note: The HPGs can be easily found in the head after removal of cuticle. The HPG is a botryoid organ that exists around the brain in the head. Remove the tracheal tissue which is a white membranous tissue under the cuticle. Then, dissect the HPGs from the head by slowly pulling them with tweezers. Classify HPGs with large and circular acini as 'Developed'. Classify HPGs with small and distorted acini as 'Shrunken'. Classify HPGs corresponding to neither 'Developed' nor 'Shrunken' as 'Intermediate'. Note: Representative photographs that show these three classes of HPG states are indicated in Figure 2. Subject 'Developed' and 'Shrunken' HPGs to RNA extraction as 'nurse bee HPGs' and 'forager HPGs', respectively, and compare gene expression in the HPGs between nurse bees and foragers (section 4). 2. Injection of 20E Collect nurse bees from typical colonies as described in the procedure 1.6.1. Note: HPGs cannot be examined in these bees as the bees will be reared. Anesthetize the bees at 4 °C in a refrigerator (not on ice). Note: It takes about 30 min to achieve anesthesia. If the anesthesia time is shortened, the number of bees in the cage should be reduced to 1-3 bees per cage. Using tweezers, immobilize the bees on dental wax placed on a Petri dish. Inject 1 µl of 20E solution into the anterior aspect of the head using an injection tip made from a calibrated capillary micropipette using a glass needle puller. Dissolve 20E in ethanol-insect saline (130 mM NaCl, 5 mM KCl, 1 mM CaCl2) mixture (1:4) at 2.5 µg/µl. Connect the injection tip to a rubber tube, and connect a yellow micropipette tip to the opposite side of the tube. Place 1 µl of 20E solution on parafilm using a micropipette, hold the yellow micropipette tip in the mouth, and draw the solution into the injection tip. Insert the injection tip into the base of the antennae, and inject the solution by blowing through the yellow micropipette tip. Note: The protocol shown in this step is the representative injection method, but the use of the automated injection machine is recommended for safety as necessary20. Rear the injected bees in insect cages at 33-36 °C (the ideal temperature is 35 °C) under dark and humid conditions (>60 % relative humidity) for 1 or 3 days. Supply honey-water mixture (1:1) to the bees. If needed, count surviving bees after injection. 3. Application of Methoprene Collect the combs that contain pupae from typical colonies after all adherent adult bees are removed with a brush. Incubate the combs at 33-36 °C (the ideal temperature is 35 °C) in humid conditions (>60% relative humidity) for up to 1 day. Collect newly emerged bees from the collected combs and apply paint marks to their thoraces using poster paint as described in the protocol for preparing single-cohort colonies. Return the marked bees to their colonies. After 6 days, collect painted workers (6 day old) from the colonies. Pick up painted workers from the combs by holding their thoraces with tweezers. Anesthetize the bees at 4 °C in a refrigerator (not on ice) as in the procedure 2.2. Using tweezers, immobilize the bees on dental wax placed on a Petri dish and apply 1-5 µl of methoprene solution (50-250 µg/µl) dissolved in acetone to their heads using a micropipette. Use a filter tip that is resistant to acetone. Note: Acetone may have the harmful effects on bees leading to death. If the high mortality of bees is observed, the reduction of the volume of acetone to 1 µl at the minimum is recommended. Rear the bees in insect cages at 33-36 °C (the ideal temperature is 35 °C) for 7 days under dark and humid conditions (>60% relative humidity). Supply honey-water mixture (1:1) to the bees. If needed, count surviving bees after topical application. Note: In this article, surviving bees are counted 7 days after application (Table 3). 4. RNA Extraction and Quantitative RT-PCR Anesthetize bees and dissect HPGs as described in steps 1.7.1 to 1.7.4. Collect the dissected HPGs in a microcentrifuge tube on dry ice and store at -80 °C until use. Add 400 µl of the reagent for protein denaturation. Note: This reagent is commercially available (see the Table of Materials). Homogenize the HPG tissues using a hand held electric mixer with a homogenization pestle that fits inside a microcentrifuge tube. Homogenize at a rotation speed of approximately 300 rpm for 1 min on ice to break and lyse tissue cells. Note: The hand held electric mixer is commercially available (see the Table of Materials). Add 80 µl chloroform and mix well. Incubate for 5 min at RT. Centrifuge the microcentrifuge tubes at 14,170 x g for 15 min at 4 °C. Transfer the aqueous phase to a new tube. Add an equal volume of isopropanol and 1 µl glycogen (5 mg/ml). Incubate at -20 °C for at least 20 min. Centrifuge the microcentrifuge tubes at 14,170 x g for 10 min at 4 °C and remove the supernatant. Add 400 µl of 75% ethanol to the pellet and mix well, centrifuge the microcentrifuge tubes at 14,170 x g for 15 min at 4 °C, and remove the supernatant. Repeat these procedures once more. Air-dry the pellet for 5-10 min and then dissolve it in 10 µl RNase-free water. Note: Complete drying of the pellet may cause insolubilization of the RNA pellet in water. Thus, the pellet which is slightly wet is appropriate for dissolving. Measure the RNA concentration using a spectrophotometer such as NanoDrop or similar. Treat 500 ng of total RNA with 2.0 U of DNase I by incubating at 37 °C for 30 min to degrade any contaminating genomic DNA. Reverse-transcribe 200 ng of DNase I-treated RNA and perform real time PCR using commercially available kits (see the list in the Table of Materials and Reagents) and gene-specific primers (mrjp2; 5'-AAATGGTCGCTCAAAATGACAGA-3', and 5'-ATTCATCCTTTACAGGTTTGTTGC-3', Hbg3; 5'-TACCTGGCTTCGTGTCAAC-3' and 5'-ATCTTCGGTTTCCCTAGAGAATG-3'). Perform the PCR as follows: [95 °C x 30 sec + (95°C x 5 sec + 60°C x 15 sec + 72 °C x 20 sec) x 45-55 cycles]. Normalize the amount of transcript with that of elongation factor 1α-F2 (EF1α˗F2) or ribosomal protein 49 (rp49)9,16,21,22. These genes are reliable control genes for analyzing gene expression in the HPGs using qRT-PCR. Perform two-tailed t-test to detect the significant differences between two experiment groups (nurse bees vs. precocious foragers, 20E-treated bees vs. control bees, and methoprene-treated bees vs. control bees) using statistical software. Note: If F-test assumes the homogeneity of variance, Student's t-test can be used. If not, Welch's t-test can be used.

Representative 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

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