This technique serves as a foundational method for experiments designed to assay the embryonic or larval development of the cricket. The main advantage of this technique is that it is flexible and will support several types of experimental approaches, such as those utilizing RNA interference or genomic manipulation. Demonstrating the procedure will be Hadley Wilson Horch, the principal investigator in the laboratory.
To begin this procedure wet the spinning grinder plane of the beveler with dripping water. Place the pulled needle into the beveler and turn it to a 20 degree angle. Lower the needle until it just touches the grinding plate and bevel it for two to three minutes.
Assess the bevel by placing the beveled needle on double stick tape that has been adhered to a glass microscope slide. Place the slide on the stage of a compound microscope equipped with a camera and image acquisition software. Acquire an image of the needle using a 20x objective.
Use the imaging software to measure the interior lumen diameter of the needle just proximal to the beveled opening. Discard any needles with an opening smaller than eight micrometers or larger than 12 micrometers. First, make 40 milliliters of 1%agarose in water and pour it into a 10 centimeter Petri dish.
Place an egg well stamp on the surface of the agarose before it solidifies. Once the agarose is solidified, remove the stamp to reveal the wells. Next, fill the agarose well plate with HBS containing 1%penicillin streptomycin.
Place the lid on the dish, wrap it in Parafilm and store it at four degrees Celsius. After this, fill a 35 millimeter Petri dish with white playground sand to make an egg collection dish for the crickets to lay eggs in. Cover the egg dish with a paper towel square cut to approximately 18 by 18 centimeters.
Fill with tap water through the paper towel. Then, tilt the Petri dish and gently squeeze the top to remove excess water. Tuck the corners of the paper towel square under the dish and place the egg dish in an inverted lid which will help keep the paper towel in place.
Place the egg dish into the cricket bin and allow the adult females to oviposit the eggs for one to two hours. Meanwhile, allow the agarose dish to warm to room temperature in preparation for holding the eggs for injection. When ready, remove the egg collection dish, now containing freshly laid eggs, from the cricket bin.
Remove the paper towel and place a strainer with a pore size between 0.5 and one millimeters over a beaker. Rinse the contents of the egg laying dish into the strainer under gently running tap water. The sand grains will fall through the strainer mesh into the beaker below while the cricket eggs remain in the strainer basket.
Fill a container with reverse osmosis 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.
Next, use scissors to cut the tip off a P1000 pipette tip to make an opening approximately three millimeters in diameter. Place this tip on a P1000 pipetter, and use it to transfer the eggs from the container to the agarose egg molds dish. Using plastic tweezers, line up the eggs in the agarose wells.
Each egg will sink to the bottom of an individual well. Cover the Petri dish with a lid until ready to inject. To begin, place the dish of eggs under the dissecting microscope and select a low magnification of around 10x.
Draw up 1.5 microliters of injection solution using 20 microliter loading tips and a P10 pipette and insert the loading tip into the wide end of the injection needle. Expel the injection solution into the injection needle. Insert the needle into the injection housing and tighten, making sure that the needle is properly and firmly inserted into the housing.
Then carefully insert the injection housing into the micromanipulator, being careful to not break or be injured by the needle. While looking at both the eggs and needle through the microscope, move the needle near the X in the top left corner of the dish grid. Lower the needle until the tip enters the HBS plus penicillin streptomycin buffered solution in the dish.
Center the needle in the field of vision and move the egg dish so that the needle is a few millimeters closer to the edge of the dish than the eggs. After this, set the microscope to the filter appropriate for Rhodamine to observe the fluorescence in the needle and focus on its tip. On the microinjector slowly turn the balance knob clockwise until the injection solution starts to leak out of the needle into the suspension solution.
Then turn the knob back counterclockwise slightly until the dye just stops leaking out of the needle. With the needle still centered in the field of view, move the egg dish so that the needle is aimed at the egg to be injected first. Adjust the magnification to about 50x so that a single egg fills most of the field of view.
Use both the micromanipulator and one's hand on the egg dish to move the needle into position for the injection. Use the micromanipulator to advance the needle and insert the tip into the first egg to be injected, making sure to insert the needle at 20 to 30%of the egg length from the posterior end of the egg perpendicular to the long axis of the egg. Inject the solution with either 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. After this, 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 the well while retracting the needle.
In this study, cricket eggs are injected using a technique that serves as a foundational method in many experiments, including but not limited to, RNA interference and genomic manipulation. The survival rate is one important parameter for assessing the success of this experiment. Most dead eggs can easily be identified by sight as the yolk and embryonic tissue within the egg begin to clump unevenly, and eventually most of the yolk will migrate to one side of the egg.
When assessed after four to six days, greater than 85%of uninjected eggs survived, while eggs injected with experimental reagents generally had a lower survival rate. Controlling for all aspects of the injection itself, including the needle puncture, the introduction of dye and buffer, or the presence of vehicle or double stranded RNA, is required in order to understand the true effects of the experimental manipulation attempted. The way one assesses the phenotypic results of injection depends on what was injected.
For example, phenotypic changes as a result of specific mRNA knockdowns may be obvious at the gross anatomy level. If on the other hand a cDNA encoding an EGFP tagged histone 2B gene under the control of a G.bimaculatus actin promoter is inserted into the cricket genome using piggyback transposase, it becomes possible to visualize each nucleus in the egg using fluorescence to excite the EGFP tagged hist 2B protein. Adjusting the balance so that the needles don't clog is a critical step in this procedure.
You may need to make several adjustments after subsequent injections since yolk sometimes clogs the needle tip. The cricket is a hemimetabolous insect that branches basally to well studied holometabolous insects such as Drosophila. Studying cricket development will help us understand more about evolution and development across the animal kingdom.
This technique takes some practice. We recommend practicing control injections until the survival rate four or five days after injection is at least 80%
