Injecting Gryllus bimaculatus Eggs

Summary

Here we present a protocol to inject cricket eggs, a technique which serves as a foundational method in many experiments in the cricket, including, but not limited to, RNA interference and genomic manipulation.

Abstract

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been developed in traditional model systems, it is difficult to manipulate genes or messenger RNA (mRNA) in most other organisms. At the same time, evolutionary and comparative approaches rely on an exploration of gene function in many different species, necessitating the development and adaptation of techniques for manipulating expression outside currently genetically tractable species. This protocol describes a method for injecting reagents into cricket eggs to assay the effects of a given manipulation on embryonic or larval development. Instructions for how to collect and inject eggs with beveled needles are described. This relatively straightforward technique is flexible and potentially adaptable to other insects. One can gather and inject dozens of eggs in a single experiment, and survival rates for buffer-only injections improve with practice and can be as high as 80%. This technique will support several types of experimental approaches including injection of pharmacological agents, in vitro capped mRNA to express genes of interest, double-stranded RNA (dsRNA) to achieve RNA interference, use of clustered regularly interspaced short palindromic repeats (CRISPR) in concert with CRISPR-associated protein 9 (Cas9) reagents for genomic modification, and transposable elements to generate transient or stable transgenic lines.

Introduction

The ability to modify the genome or influence gene expression in organisms is the basis for the design of many types of experiments testing functional causality. It is also critical for the comparative and evolutionarily-relevant work that genomic and non-genomic modification techniques be available in organisms outside traditional genetic laboratory animal model systems (e.g., Mus musculus, Danio rerio, Drosophila melanogaster, and Caenorhabditis elegans). Whether it is the desire to understand organismal diversity¹ or one's adherence to Krogh's principle, that for every biological question there is an organism best suited to its solution^2,3, the ability to modify genomes or influence the gene expression is essential for modern experimental designs.

The cricket Gryllus bimaculatus is an emerging model system. Used for the last century in neuroethology experiments⁴, the last two decades have witnessed an increased experimental interest in the cricket, particularly focused on the evolution and development of this organism⁵. The cricket is a hemimetabolous insect that branches basally to well-studied holometabolous insects, such as D. melanogaster and Tribolium castaneum⁶. Due to its useful position on the evolutionary tree, scientists are interested in asking modern, sophisticated experimental questions in this insect, which has led to a growing interest in adapting molecular tools for the use in G. bimaculatus.

Injections of molecular reagents into cricket eggs can be used for genomic modification experiments as well as non-genomic manipulations of gene expression in embryos. For example, transgenic G. bimaculatus carrying eGFP insertions have been created using the transposase piggyBac^7,8. Investigators have successfully created knockout G. bimaculatus using Zinc-finger nucleases (ZFNs) and transcription activator-like (TAL) effector nucleases (TALENs) to introduce double-stranded breaks in specific genomic regions⁹. Though ZFNs and TALENs allow site-specific targeting in animals beyond the big four model systems, these reagents have quickly been surpassed by the CRISPR/Cas9 system, which is simpler to use, more efficient, and highly flexible¹⁰. CRISPR has been used in G. bimaculatus to produce knock-out¹¹ as well as knock-in lines^12,13 In addition to genomic modification, dsRNA can be injected into eggs to knock down mRNA expression in developing embryos, allowing investigators to understand the role of specific transcripts throughout development^14,15. Some limited details on how to inject cricket eggs have been published previously¹².

Here, we describe a detailed protocol for injecting early G. bimaculatus eggs. This protocol is effective and easily adaptable to various laboratory settings, injection materials, and possibly to other insects. While additional details for designing and implementing genomic modification and knockdown experiments have been published elsewhere^12,13, these approaches will ultimately rely on the injection protocol detailed here.

Protocol

1. Hardware Setup and Preparation of Materials

NOTE: Please see Table 1 and Table of Materials for preparation of solutions, reagent, and equipment details.

1. Set up a dissecting microscope in order to see eggs and guide the injection needle. (Figure 1A shows a dissecting microscope equipped with fluorescence.) Fluorescence capability is advantageous but not necessary.
2. Position a 3-axis micromanipulator to maneuver the injection needle into place (Figure 1A).
3. Set up a microinjector with tubing and a needle holder to inject small amounts of material (Figure 1A). Optionally use a foot pedal which is not shown in the figure.
4. Design and create a stamp to create wells for eggs (Figure 1C) by printing the inverse of the desired pattern, either with a 3D printer or a laser engraver.
   NOTE: The 3D printed sample stamp shown is 4.5 cm x 5 cm with 128, 3 cm x 1 cm x 1 mm protrusions for creating individual wells. Details on how to make a variety of customizable molds have been published elsewhere¹⁶.
5. Prepare 10x Injection Buffer, HEPES buffered saline (HBS), and a stock solution of Tetramethylrhodamine Dextran dye and store at 4 °C.
6. Prepare experimental solutions to be injected, such as dsRNA, CRISPR/Cas9¹², transposable elements, in vitro capped mRNA^7,8, pharmacological agents¹⁷, ZFNs, or TALENS⁹.
7. Place double-stick tape on several layers of standard lab tape on a glass microscope slide in order to create a platform 1 mm or so above the glass slide, which will be used for holding needles for the assessment.
8. Prepare a cricket container approximately 40 cm x 60 cm in size with a lid possessing a mesh-covered opening for air circulation.
9. Add food, water, and sheltering materials.
10. Add several dozen healthy male and female adult crickets. Identify adult cricket by the presence of wings.
    NOTE: A range of post-imaginal ages may increase egg yields. The density of crickets should be relatively high in order to be able to collect enough eggs for an experiment, but not so crowded that cannibalism occurs. For example, approximately two dozen healthy crickets, with a roughly equal male to female ratio, in a container of the size noted above should be sufficient to collect about 200 eggs in a one to two h period. Crickets can be kept at room temperature, but development and behavior will be optimal if crickets are maintained in a warm (23.5–26 °C), humid (40 - 60% relative humidity) incubator.

2. Making Needles for Injection

1. Use Borosilicate Glass Capillaries with filament (see the Table of Materials for size details). Prepare the pull needles on the same day, or up to a few days before injections are completed.
2. Place a capillary glass into the puller, center the glass on the filament, and tighten the knobs to secure the glass in place.
3. Set the puller temperature near the glass ramp temp (-5 °C to +10 °C).
4. Use a single-step, high-heat protocol to pull a long taper with a small opening but avoid melting the ends closed.
   NOTE: For example, when using the micropipette puller detailed in the Table of Materials equipped with a box filament, use the following parameters for glass with a ramp temperature of 478: Heat 478; Pull 45; Vel 75; Del 120. Optimal conditions for producing a needle with the shape shown in Figure 2B should be determined empirically by each user.
5. Bevel needle tips in preparation for the injections.
6. Wet the spinning grinder plane of the beveler (Figure 2A) with dripping water. Use either reverse osmosis (RO) or diethylpyrocarbonate (DEPC)-treated distilled water.
7. Place the pulled needle into the beveler and turn to a 20° angle.
8. Lower the needle until it just touches the grinding plane and bevel for 2 to 3 min.
9. Assess the bevel by placing the beveled needle on double-stick tape that has been adhered to a glass microscope slide.
10. Place the slide holding the needle on the stage of a compound microscope equipped with a camera and image acquisition software.
11. Acquire an image of the needle tip using a 20x objective.
12. Use the imaging software to measure the interior lumen diameter of the needle just proximal to the beveled opening (Figure 2C). The optimal size is 10 µm + 2 µm.
13. Discard needles that have an opening below 8 µm and above 12 µm.
14. Store the needles in a container with a lid to avoid getting dusty. Use a strip of wax or similar material to elevate the needles off the bottom of the container to prevent breaking the tips (Figure 2D).

3. Egg Collection and Preparation

1. Maximize the number of eggs laid by depriving crickets of any moist laying material (water vials, egg dishes) overnight or for at least 8–10 h before attempting to collect eggs.
   NOTE: Since females have the ability to internally store sperm, a differently timed procedure may be necessary if carefully timed fertilization is experimentally essential¹⁸.
2. Make 40 mL of 1% agarose in water and pour into a 10 cm petri dish.
3. Place an egg-well stamp on the surface of the agarose before it solidifies.
4. Remove the stamp, once the agarose is solidified, to reveal the wells (Figure 1C).
5. Fill the agarose well plate with HBS containing 1% Penicillin/Streptomycin.
6. Place the lid on the dish, wrap in parafilm and store at 4 °C.
   NOTE: Dishes can be stored for a few days at 4 °C but should be warmed up to room temperature before use to avoid condensation.
7. Make an egg collection dish for crickets to lay eggs in by filling a 35 mm petri dish with white playground sand (Figure 1B).
   NOTE: The sand specified in the Table of Materials is of the appropriate grain size. If the sand grains are too large, the sand will damage the eggs.
8. Cover the egg dish with a square of paper towel cut to be approximately 18 cm x 18 cm, and place on the top of dish with sand. Through this paper towel cover the filled dish with tap water.
9. Tilt the petri dish and gently squeeze the top to remove excess water.
10. Tuck the corners of the paper towel square under the dish and place the egg dish in the inverted lid, which will help keep the paper towel cover in place.
11. Place the egg dish into the cricket bin and allow the adult females to oviposit eggs for one to two h.
    NOTE: The relatively short time here ensures that all the eggs will be similar in the stage of development at the time of injection. If injection before cellularization is important, injections should occur within 14 h of egg laying¹⁹.
12. Meanwhile, allow the agarose dish (from step 3.6) to warm to the room temperature in preparation for holding eggs for injection.
13. Remove egg collection dish, now containing freshly laid eggs, from the cricket bin, and remove the paper towel cover. Place a strainer with a pore size of 0.5–1 mm over a beaker of at least 500 mL capacity (Figure 1B).
14. Rinse the contents of the egg-laying dish (sand and eggs) into the strainer under gently running tap water letting the sand grains fall through the strainer mesh into the water in the beaker below but leaving the cricket eggs in the strainer basket.
15. Fill a container with RO water and place it in a tray. Invert the strainer over the container and tap it against the dish to dislodge the eggs into the water. The eggs will sink to the bottom of the container.
16. Cut the tip off a P1000 pipette tip with scissors to make an opening approximately 3 mm in diameter. Place this tip on a P1000 pipettor and use it to transfer the eggs from the container to the agarose egg mold dish, transferring as little water as possible.
17. Use plastic tweezers to line up eggs in agarose wells filled with HBS plus 1% Penicillin/Streptomycin. Each egg will sink to the bottom of an individual well. Cover with the petri dish lid until ready to inject.

4. Prepare the Microinjector

1. Attach the microinjector to a compressed air or gas source (this protocol was optimized using either compressed air or N₂).
   NOTE: If using a portable air tank, fill to at least 75 psi. The tank will lose air pressure throughout the injections and may need to be refilled; injections become difficult below 40psi.
2. Turn on the microinjector. Set the injection time to 0.17 s and the pressure to 10–15 psi.
   NOTE: The exact pressure needed will depend on the opening of the needle and must be determined empirically for every round of injection and/or new needle used.
3. Ensure that the BALANCE knob of the microinjector is turned to 0 (typically fully counter-clockwise) and that the microinjector is pressurized and in the injection mode.

5. Injections

1. Filter about 500 µL of 10x Injection Buffer using a 1 mL syringe and a 0.45 µm syringe filter.
2. Mix injection reagents (the details that follow are for the injection of dsRNA prepared as described in¹²).
   NOTE: It may be advisable to design and perform several controls, especially when one is first learning this technique. Poking eggs without injecting, injecting buffer + dye alone, or injecting buffer + dye + a control reagent (such as eGFP dsRNA) are all good tests of how disruptive various elements of the injection protocol might be. See Figure 3 for the survivability of a number of different controls.
3. Start with a 4 μL dsRNA aliquot.
4. Add 0.5 μL of the filtered 10x Injection Buffer to the dsRNA aliquot.
5. Add 0.5 μL of 50 mg/mL Tetramethyl rhodamine Dextran dye stock solution. If working on a dissecting scope without fluorescence, use Phenol Red instead.
6. Keep all materials on ice for the duration of the injection period.
7. Load the injection solution into an injection needle using 20 μL loading tips and a p10 pipet.
8. Draw up a 1.5 μL injection solution and insert the loading tip into the wide end of the injection needle.
9. Eject solution as far down into the needle as possible and eliminate air bubbles by flicking gently; be careful not to break the needle.
10. Place the dish of eggs under the dissecting microscope and select a low magnification of around 10x (1x magnification viewed through 10x eye objectives).
11. Insert the needle into the injection housing and tighten.
    NOTE: Take care that the needle is properly and firmly inserted into the housing. To do this, pull the wide end of the needle back out of the metal housing, bringing the silicon tubing in the housing with it. Adjust the silicon tubing so that the glass end of the needle protrudes just beyond the silicon. If this is not done, the silicon tubing may get pinched between the glass and the metal housing, blocking the end of the needle.
12. Carefully insert the injection housing with an attached needle into the micromanipulator. Be aware of the sharp end of the needle and be careful that it is not bumped into surfaces and broken.
13. While looking at both the eggs and the needle through the microscope, move the needle near the eggs in the top left corner of the dish grid.
14. Lower the needle until the tip enters the HBS plus 1% Penicillin/Streptomycin buffered solution in the dish.
15. Center the needle in the field of vision and move the egg dish so that the needle is a few mm closer to the edge of the dish than the eggs.
16. Do not obstruct the view of the grid of eggs with the needle.
17. Set the microscope to the filter appropriate for Rhodamine so it is possible to observe the fluorescence in the needle and focus on the tip of the needle.
18. On the microinjector, slowly turn the BALANCE knob clockwise until the injection solution starts to leak out of the needle into the suspension solution. The balance number on the screen of the microinjector will begin to increase, though the numerical value is not important. Next, turn the knob back counterclockwise slightly just until the dye stops leaking out of the needle.
    NOTE: It is critical to set this balance each time a new needle is used. Without the balance set appropriately, the microinjector will continue to inject for some time after the injection is triggered. It is even possible that a single push of the injection button with an unbalanced needle will result in the expulsion of the entire contents of the loaded needle.
19. With the needle centered in the field of view, move the egg dish so that the needle is aimed at the egg to be injected first. It is recommended that injections start in one corner of the grid of arranged eggs, for example, the upper left corner.
20. Adjust the magnification to about 50x; at this magnification, a single egg will fill most of the field of view.
21. Use both the micromanipulator and one’s hand on the egg dish to move the needle into position for the injection.
22. Advance the needle using the micromanipulator and insert the tip of the needle into the first egg to be injected.
23. Insert the needle at 20–30% egg length from the posterior (blunter) end of the egg (Figure 1E), perpendicular to the long axis of the egg.
24. Inject solution either with the injection foot pedal or the injection button on the microinjector. A small bolus of fluorescent material inside the egg will indicate a successful injection (Figure 1D).
    NOTE: It is not uncommon for some yolk to be ejected from the egg during injection (Figure 1F), and this does not necessarily indicate that the injected egg will be inviable.
25. Retract the needle from the egg. If the egg is unintentionally lifted out of its well upon retraction of the needle, use small forceps to push the egg back into its well while retracting the needle.
26. If the needle clogs during injection, remove the needle from the egg and, keeping the needle submerged in the HBS plus 1% Penicillin/Streptomycin solution covering the eggs, clear the clogged needle using either the Clear function or by pushing the inject button.
27. Continuing to use the same needle, repeat the injection procedure for as many eggs as possible. At first, this may be only about 20 or 30 eggs but will increase to over 100 with practice.
28. If the needle becomes clogged and cannot be cleared, discard the needle, fill a new needle and begin again (i.e., return to step 5.7).
29. Store the dish of injected eggs in a warm incubator (23.5–26 °C) that is slightly humid (to minimize the evaporation) until the embryos have reached the desired stage of development for the analysis.
30. At least every 24 h, remove eggs that are no longer viable (Figure 3A,B), and replace the suspension solution with fresh HBS plus 1% Penicillin/Streptomycin.
31. Two to three days after injection, transfer the eggs by gentle pipetting or with plastic forceps to paper towels moistened with HBS plus 1% Penicillin/Streptomycin in a lidded petri dish to continue development in the warm, humid incubator.
32. Monitor the eggs, and remove eggs that are no longer viable each day (Figure 3A,B).

Results

Crickets readily lay eggs in the moist material, and providing adequate material, such as moist sand or dirt, induces them to lay a large number of eggs. This is especially effective if crickets are first deprived of egg-laying material for 8–10 h. Eggs laid in clean sand can be easily separated, collected (Figure 1B) and placed into custom-designed egg wells for injection (Figure 1C). A dissecting microscope, a microinject...

Discussion

The two main challenges with this technique are the related issues of optimal needle size and survivability. Though smaller needles improve survivability, needles with narrower lumens have a greater degree of capillary forces at work, which makes it more likely that yolk will move into the needle causing it to clog. In the best case, blockages can be cleared simply by injecting another egg or by clearing the needle as described above. One can also attempt to increase the balance pressure on the microinjector until the yo...

Materials

Name	Company	Catalog Number	Comments
Fluorescent dissecting microscope	Leica	M165 FC	Stereomicroscope with fluorescence
External light source for fluorescence	Leica	EL 6000
Microinjector	Narishige	IM-300	-Accessories may include Injection Needles Holder, Input Hose (with a hose connector), AC Power Cord, Foot Switch, Silicone Rubber Gasket-
mCherry filter cube	Leica	M205FA/M165FC	Filter cube for mCherry or similar red dye will work
Micromanipulator	World Precision Instruments, Inc.	M3301R	Used with Magnetic Stand (Narishige, Type GJ-8)
Magnetic stand	Narishige	MMO-202ND
Pipette Holder (Needle holder)	Narishige	HD-21
Tubing to connect air source to microinjector
Egg well stamp	3D printed	custom	3D printed on a Lulzbot Taz 5 using Poly Lactic Acid thermoplastic
Microwave	various
Incubator or temperature controlled room	various		Temperatures of 23.5-26 °C are needed.
Cricket food	various		cat food or fish flakes are appropriate food.
Cricket water	vairous		Water can be held in vials and presented to crickets through cotton balls
Cricket shelter	arious		Shelter materials can include crumpled paper towels or egg cartons
Glass capillary tubes	World Precision Instruments, Inc.	Item no. 1B100F-4	Kwik-Fil™ Borosilicate Glass Capillaries, 100 mm length, 0.58 mm ID, 1.0 mm OD, with filament
Micropipette puller	Flaming/Brown	Model P-97	Distributed by Sutter Instrument Co.
Beveller/Micro grinder	Narishige	Model EG-45/EG-400	EG-400 includes a microscope head
Petri dishes	CellTreat	Product code 229693	90 mm diameter
Play Sand	Sandtastik Products Ltd.	B003U6QLVS	White play sand
Agarose	American Bioanalytical	AB000972	Agarose GPG/LE ultrapure
Egg Strainer: Extra Fine Twill Mesh Stainless Steel Conical Strainers	US Kitchen Supply	Model SS-C123	Pore size should be between 0.5 - 1.0 mm
Penicillin Streptomycin	Gibco by Life Technologies	Ref 15070-063	Pen Strep
Plastic tweezers	Sipel Electronic SA	P3C-STD	Black Static Dissipative, 118 mm
Syringe filters, 25 mm diameter, 0.45 µm	Nalgene	725-2545	Use with 1 mL syringe
1 mL syringe, with Tuberculin Slip Tip	Becton Dickinson	309602	Use with syring filter to filter Injection Buffer , Luer-Lok tip syringes would also work
Air tank (optional)	Midwest Products	Air Works®	Portable air tank
Rhodamine dye	Thermofisher	D-1817	dextran, tetramethylrhodamine 10,000MW,
20 mL loading tips	Eppendorf	Order no. 5242 956.003	epT.I.P.S. 20 μL Microloader
Compound microscope	Zeiss	Axioskope 2 plus
20x objective	Ziess	Plan-Apochromat 20x/0.75 M27
Camera	Leica	DMC 5400
Leica Application Suite  software	Leica	LAS	Version 4.6.2 used here