RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees (Apis mellifera)

Summary

In this protocol, we describe two strategies that simultaneously suppress two genes (double gene knockdown) in honey bees. Then we present how to use the proboscis extension response (PER) assay to study the effect of double gene knockdown on honey bee gustatory perception.

Abstract

This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception.

RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species.

The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

Introduction

RNA interference (RNAi) is RNA based post-transcriptional gene silencing, which occurs in a wide variety of eukaryotic organisms. The process of RNAi is triggered by endogenous or exogenous double-stranded RNA (dsRNA) precursors. The dsRNA activates the ribonuclease protein Dicer which binds and cleaves the dsRNA to small fragments (20-25 bp). Then the small fragments of the dsRNA guide a recognition and cleavage of complementary mRNAs by argonaute proteins, a catalytic component of RNA-induced silencing complex (RISC)¹. In mammals, dsRNAs longer than 30 nt, activate an antiviral response (interferon response, IFN) which leads to nonspecific degradation of RNA transcripts ². However, long dsRNAs have proven to be effective and specific in insects since there is a lack of this IFN ³.

Long dsRNAs have been used for downregulation of target genes in different insect species. Honey bees are one of the pioneer insect organisms in which functions of many important genes in the development and behavior have been revealed by using dsRNA ^4,5. Several dsRNA delivery methods have been performed in honey bees: dsRNA feeding efficiently downregulates target gene expression in honey bee larvae ⁶, whereas dsRNA injection is an effective approach for gene knockdown in honey bee embryos ⁴ and adult bees ^7,8.

Gene knockdown effects exhibited by applying dsRNA to insects are transient and localized. Studies have shown that both abdominal dsRNA injections and thoracic dsRNA injections effectively suppress target gene expression in abdominal fat body cells of insects ^9,10. DsRNA is injected into abdominal and thoracic cavities and fat body cells are able to take up the dsRNA from the hemolymph where the cells are bathed ¹⁰. However, genes in other organs, such as ovaries and brains, cannot be targeted by either abdominal or thoracic injections. In order to target genes in honey bee brain, brain injection of dsRNA has also been performed, which effectively influences target gene expression in local brain areas ¹¹. Here, we only document abdominal dsRNA injection which is more commonly used in adult honey bees.

RNAi has been primarily used to target a single gene and has been a powerful tool to reveal the gene function. However, any gene is not isolated from others; it is in complex regulatory networks. A key to understand a biological process is to dissect how genes interact with each other, which requires simultaneous manipulations of multiple genes rather than a single gene knockdown. In mammalian cell lines, scientists have succeeded in simultaneously inhibiting two or three genes by using delivery systems ¹² or multi-microRNA (miRNA) hairpin designs ¹³. But in insects, multiple gene knockdowns are still untested. Here, we present different injection strategies which can achieve a double gene knockdown. We target two genes: vitellogenin (vg) which encodes a yolk protein precursor, and ultraspiracle (usp) which encodes a putative receptor for juvenile hormone (JH) and may serve as a transcription factor mediating responses to JH ¹⁴ in honey bees. Vg and JH regulate each other in a feedback loop ¹⁵ and are involved in honey bee behavioral regulation ⁹. Using the double gene knockdown, we perturb both Vg and JH pathways, and discover how they jointly affect honey bee behavior and physiology and how vg, usp and JH interact ⁹.

Gustatory perception is a behavioral predictor for honey bee social behavior ¹⁶. In terms of behavioral development, nest bees with high gustatory perception behaviorally mature fast, and usually forage early in life and prefer to collect pollen ^16,17. Although regulatory mechanisms underlying gustatory perception are still unclear, studies have shown that gustatory perception is linked to internal energy metabolisms ⁹, hormonal secretion ^18,19 and biogenetic amine pathways ²⁰. Both Vg and JH are important hormonal regulators modulating gustatory perception ^7,21. In the laboratory, a variation of gustatory perception in honey bees can be evaluated by testing the proboscis extension response (PER) to different sucrose solutions. Each bee is tested by touching both her antennae with a droplet of water followed by an ascending concentration series of 0.1, 0.3, 1, 3, 10, 30% sucrose. A positive response is noted if a bee fully extends her proboscis when a droplet of water or sucrose is touched to each antenna. Based on the number of positive responses to the solutions, the gustatory perception level of each individual can be determined ¹⁶. However, the application of the PER is not limited to measuring gustatory perception. The PER is also an effective method to test the metabolic state of bees such as satiation vs. hunger. The bees with greater responses to sucrose are hungrier in general (Wang and Amdam, unpublished data). Furthermore, the PER paradigm can also be used in associative learning and memory in honey bees. In this case bees will be trained to associate the presence of sucrose water with an odor. When the bees learn the association, only the presence of the odor can evoke a positive proboscis response without rewarding them with the sucrose ^22,23. In this video, we show how to perform PER to evaluate gustatory perception which has been connected with vg and usp double knockdown in a previous study ⁹.

Protocol

Part 1: RNAi-mediated double gene knockdown

1. dsRNA Synthesis

1. Design primers of dsRNAs targeting vg, usp and a control gene encoding green fluorescence protein (GFP) which is not in the honey bee genome: primers were designed using online free software Primer3 (http://frodo.wi.mit.edu/).
2. dsRNA synthesis for vg, usp and GFP: use RiboMax T7 RNA production system from Promega for in vitro transcription.
3. dsRNA purification:
   1. Denaturation and renaturation: heat the dsRNA to 85 °C for 5 min and let it cool down at room temperature for 1 hr.
   2. DNase I treatment: add 1 μl DNase I into each dsRNA reaction and mix them by flicking the tubes. Incubate for 15 min at 37 °C.
   3. Add 150 μl nuclease-free water and 750 μl TRizol - LS into each reaction, and gently mix them by inverting the tube. Incubate the samples for 5 min at 30 °C.
   4. Add 200 μl chloroform into each sample. Mix vigorously for 20 sec. Centrifuge the samples for 15 min at 12,000 x g at 4 °C.
   5. Transfer the supernatant phase to each new tube and add 500 μl isopropyl alcohol into each tube. Mix them by inverting the tube. Incubate the mixture for 20 min at -20 °C. Centrifuge it for 10 min at 12,000 x g at 4 °C.
   6. Remove the supernatant and use 1,000 μl 75% ethanol to wash the pellet. After centrifuging the tube for 5 min at 7,500 x g at 4 °C, air dry the dsRNA pellet, and use nuclease free water to dissolve the pellet. In order to ensure the efficacy of gene down-regulation, the dsRNA concentration should be around 9 -10 μg/μl.

2. dsRNA Abdominal Injection

For double gene knockdown, there are two strategies: 1) single injection: mix dsRNA of two genes and inject the mixture; 2) two-day injection: inject the first dsRNA targeting one gene on the first day, and inject the second dsRNA into the same bees on the second day.

1. Chill newly emerged bees in a 4 °C refrigerator for 1-2 min. When the bees are immobilized, mount 3-4 bees in parallel on Petri dishes full of solid wax with insect pins crossed between their abdomens and thoraces.
2. Chill the bees again in a 4 °C refrigerator. Ensure the bees are completely immobile. It takes about 1-2 min. The bees should look loose. If the bees become curled or contorted, it means they are chilled too long which should be avoided. Chilling too much causes high mortality, but complete immobilization is important as any movement increases the size of the wound and mortality.
3. Put a disposable 30 G needle (BD) on a Hamilton micro syringe. Take 3 μl dsRNA and make sure there is no air bubble in the syringe. The needle is inserted to a side of the abdomen to avoid damaging internal organs. Press the syringe plunger slowly to allow the dsRNA to be absorbed. Since dsRNA is very viscous, it takes 2-3 sec to be completely expelled from the syringe. After completely pushing down the plunger, leave the needle in the wound for 4-5 sec. Observe bees for 3-5 sec after injections. If a hemolymph droplet leaks from the wound, discard the bee.
4. Use different colors to mark their thoraces corresponding to different treatments. At this point if the single injection strategy was used, the bees can be placed back into a colony after 1 hr observation. However, if the two-day injection strategy was used, after being injected with the first dsRNA, the bees should be placed in a cylinder mesh cage with honey supplied on the side and be kept in an incubator at 34 °C and 80% humidity.
5. On the second day, follow steps 2.2 and 2.3 to chill the bees in the mesh cages and mount them on wax plates. Perform injections with the second dsRNA as it is described in step 2.4.
6. Let the bees recover at room temperature for 1 hr and introduce them into a colony.

Note: Knockdown effects can be detected by quantitative RT-PCR (qRT-PCR) and western blotting. In general, knockdown efficacy varies and depends on many factors. In our study, the double gene knockdown can be detected 7 days after single injections and two-day injections.

Part 2: Proboscis extension response (PER) assay

1. Collecting Bees from the Hive

On day 6 after the injections, collect treated bees from the colony without using a smoker. Collect bees in metal mesh cylinder cages. Keep 3 bees in each cage.

2. Mounting Bees for PER

Chill the bees at 4 °C until they are completely immobilized. Mount each bee vertically into a plastic tube specially designed for PER. Keep the abdomen inside the tube by using two small pieces of tape, and keep the head sticking out of the tube. Put the tubes on a rack with two rows of small plastic columns, and assign each tube (bee) with an ID number. Then put treated bees on the racks into an incubator (34 °C and 80% humidity) and keep them in the incubator for 2 hr.

3. Preparing a Series of Sucrose Water Solutions

Prepare 0%, 0.1%, 0.3%, 1%, 3%, 10% and 30% sucrose water solutions and load the solutions into syringes with 15-20 G needles.

4. PER Assay

Touch the bee's antenna with a droplet of 0% solution (water) followed by 0.1%, 0.3%, 1%, 3%, 10% and 30% sucrose water solutions. Test all bees with water first and allow at least two minutes as the interval between each solution is used. Therefore, we usually have at least 20 bees for each trial. If a bee fully extends her proboscis, a positive response is counted.

5. Data Management

Sum up the total positive responses for each individual for gustatory response score (GRS). Perform an appropriate statistical analysis on the data.

Part 3. Gene knockdown validation

Fat bodies are dissected from another set of 7-day old treated bees. A standard TRizol procedure is used for RNA extraction, which is followed by DNase I treatment ²⁴. Vg and usp gene expression is analyzed by a two-step qRT-PCR. Actin is used as a reference gene since it has stable expressions in different tissues of honey bees. Published real-time PCR primers for vg and usp gene are used in this experiment ²⁴. Data are analyzed by using Delta-Delta CT method ²⁵.

Results

Both the single-injection and two-day-injection strategies significantly reduced vg (One-way ANOVA, p < 0.001) (Figure 2A) and usp (One-way ANOVA, p < 0.001) (Figure 2B) transcript levels in honey bees six days after the dsRNA was injected. The suppression of usp transcript by using the single injection with dsRNA mixture and the two-day injection with vg first and usp second (vg/usp) was significantly lower than the tw...

Discussion

Our study is the first effort to simultaneously knock down two genes in adult insects. Our results show that the two strategies of dsRNA injections (single injection and two-day injection) are effective for the double gene suppression, and simultaneous gene silencing of vg and usp can be tested 6 days after the dsRNA injections into bees ^8,9. However, gene knockdown efficacy varies in different insect species depending on transcript level of target gene, protein turnover rates and dsRNA uptak...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
GE Healthcare PCR beads in 96 well plate	GE Healthcare	27-9557-01
QIAquick PCR purification kit	Qiagen	28104
RiboMax T7 RNA production system	Promega	P1300
Trizol LS	Invitrogen	10296028
Chloroform:Isoamyl alcohol 24:1	Sigma-Aldrich	C0549
isopropyl alcohol	Sigma-Aldrich	I9516
Hamilton micro syringe	Hamilton	80301
30G BD disposable needles	BD Biosciences	305106
Sucrose	Sigma-Aldrich	84097
1 ml Syringe	BD Biosciences	30960
Stereo dissection microscope	Leica Microsystems	S6D
Insect pins	Fine Science Tools	26000-25
Wax plate