Research reported in this project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM10342 to HH3, and by NSF award number IOS-1257217 to CGE.

1. Hardware Setup and Preparation of Materials
NOTE: Please see Table 1 and Table of Materials for preparation of solutions, reagent, and equipment details.
<ol>
	<li>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.</li>
	<li>Position a 3-axis micromanipulator to maneuver the injection needle into place (Figure 1A).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Prepare 10x Injection Buffer, HEPES buffered saline (HBS), and a stock solution of Tetramethylrhodamine Dextran dye and store at 4 &#176;C.</li>
	<li>Prepare experimental solutions to be injected, such as dsRNA, CRISPR/Cas912, transposable elements, in vitro capped mRNA7,8, pharmacological agents17, ZFNs, or TALENS9.</li>
	<li>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.</li>
	<li>Prepare a cricket container approximately 40 cm x 60 cm in size with a lid possessing a mesh-covered opening for air circulation.</li>
	<li>Add food, water, and sheltering materials.</li>
	<li>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&#8211;26 &#176;C), humid (40 - 60% relative humidity) incubator.</li>
</ol>
2. Making Needles for Injection
<ol>
	<li>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.</li>
	<li>Place a capillary glass into the puller, center the glass on the filament, and tighten the knobs to secure the glass in place.</li>
	<li>Set the puller temperature near the glass ramp temp (-5 &#176;C to +10 &#176;C).</li>
	<li>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.</li>
	<li>Bevel needle tips in preparation for the injections.</li>
	<li>Wet the spinning grinder plane of the beveler (Figure 2A) with dripping water. Use either reverse osmosis (RO) or diethylpyrocarbonate (DEPC)-treated distilled water.</li>
	<li>Place the pulled needle into the beveler and turn to a 20&#176; angle.</li>
	<li>Lower the needle until it just touches the grinding plane and bevel for 2 to 3 min.</li>
	<li>Assess the bevel by placing the beveled needle on double-stick tape that has been adhered to a glass microscope slide.</li>
	<li>Place the slide holding the needle on the stage of a compound microscope equipped with a camera and image acquisition software.</li>
	<li>Acquire an image of the needle tip using a 20x objective.</li>
	<li>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 &#181;m + 2 &#181;m.</li>
	<li>Discard needles that have an opening below 8 &#181;m and above 12 &#181;m.</li>
	<li>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).</li>
</ol>
3. Egg Collection and Preparation
<ol>
	<li>Maximize the number of eggs laid by depriving crickets of any moist laying material (water vials, egg dishes) overnight or for at least 8&#8211;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.</li>
	<li>Make 40 mL of 1% agarose in water and pour into a 10 cm petri dish.</li>
	<li>Place an egg-well stamp on the surface of the agarose before it solidifies.</li>
	<li>Remove the stamp, once the agarose is solidified, to reveal the wells (Figure 1C).</li>
	<li>Fill the agarose well plate with HBS containing 1% Penicillin/Streptomycin.</li>
	<li>Place the lid on the dish, wrap in parafilm and store at 4 &#176;C. 
	NOTE: Dishes can be stored for a few days at 4 &#176;C but should be warmed up to room temperature before use to avoid condensation.</li>
	<li>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.</li>
	<li>Cover the egg dish with a square of paper towel cut to be approximately 18 cm x 18&#160;cm, and place on the top of dish with sand. Through this paper towel cover the filled dish with tap water.</li>
	<li>Tilt the petri dish and gently squeeze the top to remove excess water.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Meanwhile, allow the agarose dish (from step 3.6) to warm to the room temperature in preparation for holding eggs for injection.</li>
	<li>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&#8211;1 mm over a beaker of at least 500 mL capacity (Figure 1B).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Prepare the Microinjector
<ol>
	<li>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.</li>
	<li>Turn on the microinjector. Set the injection time to 0.17 s and the pressure to 10&#8211;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.</li>
	<li>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.</li>
</ol>
5. Injections
<ol>
	<li>Filter about 500 &#181;L of 10x Injection Buffer using a 1 mL syringe and a 0.45 &#181;m syringe filter.</li>
	<li>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.</li>
	<li>Start with a 4 &#956;L dsRNA aliquot.</li>
	<li>Add 0.5 &#956;L of the filtered 10x Injection Buffer to the dsRNA aliquot.</li>
	<li>Add 0.5 &#956;L of 50 mg/mL Tetramethyl rhodamine Dextran dye stock solution. If working on a dissecting scope without fluorescence, use Phenol Red instead.</li>
	<li>Keep all materials on ice for the duration of the injection period.</li>
	<li>Load the injection solution into an injection needle using 20 &#956;L loading tips and a p10 pipet.</li>
	<li>Draw up a 1.5 &#956;L injection solution and insert the loading tip into the wide end of the injection needle.</li>
	<li>Eject solution as far down into the needle as possible and eliminate air bubbles by flicking gently; be careful not to break the needle.</li>
	<li>Place the dish of eggs under the dissecting microscope and select a low magnification of around 10x (1x magnification viewed through 10x eye objectives).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Lower the needle until the tip enters the HBS plus 1% Penicillin/Streptomycin buffered solution in the dish.</li>
	<li>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.</li>
	<li>Do not obstruct the view of the grid of eggs with the needle.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Adjust the magnification to about 50x; at this magnification, a single egg will fill most of the field of view.</li>
	<li>Use both the micromanipulator and one&#8217;s hand on the egg dish to move the needle into position for the injection.</li>
	<li>Advance the needle using the micromanipulator and insert the tip of the needle into the first egg to be injected.</li>
	<li>Insert the needle at 20&#8211;30% egg length from the posterior (blunter) end of the egg (Figure 1E), perpendicular to the long axis of the egg.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Store the dish of injected eggs in a warm incubator (23.5&#8211;26 &#176;C) that is slightly humid (to minimize the evaporation) until the embryos have reached the desired stage of development for the analysis.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Monitor the eggs, and remove eggs that are no longer viable each day (Figure 3A,B).</li>
</ol>

<ol>
	<li>Abzhanov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+we+there+yet?+Tracking+the+development+of+new+model+systems.">Are we there yet? Tracking the development of new model systems.</a> Trends in Genetics. 24 (7), 353-360 (2008).</li><li>Krebs, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+August+Krogh+principle:+&#8220;For+many+problems+there+is+an+animal+on+which+it+can+be+most+conveniently+studied.">The August Krogh principle: &#8220;For many problems there is an animal on which it can be most conveniently studied.</a> Journal of Experimental Zoology Part B: Molecular and Developmental Evolution. 194 (1), 221-226 (1975).</li><li>Krogh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Progress+of+Physiology.">The Progress of Physiology.</a> American Journal of Physiology. 90 (2), 243-251 (1929).</li><li>Huber, F., Moore, T. E., Loher, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cricket neurobiology and behavior. , (1989).</li><li>Horch, H. W., Mito, T., Popadic, A., Ohuchi, H., Noji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Cricket as a Model Organism: Development, Regeneration, and Behavior. , (2017).</li><li>Misof, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenomics+resolves+the+timing+and+pattern+of+insect+evolution.">Phylogenomics resolves the timing and pattern of insect evolution.</a> Science. 346 (6210), 763-767 (2014).</li><li>Nakamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+transgenic+cricket+embryos+reveals+cell+movements+consistent+with+a+syncytial+patterning+mechanism.">Imaging of transgenic cricket embryos reveals cell movements consistent with a syncytial patterning mechanism.</a> Current Biology. 20 (18), 1641-1647 (2010).</li><li>Shinmyo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac-mediated+somatic+transformation+of+the+two-spotted+cricket,+Gryllus+bimaculatus.">piggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus.</a> Development, growth &amp; differentiation. 46 (4), 343-349 (2004).</li><li>Watanabe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-transgenic+genome+modifications+in+a+hemimetabolous+insect+using+zinc-finger+and+TAL+effector+nucleases.">Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases.</a> Nature communications. 3, 1017 (2012).</li><li>Wang, H., La Russa, M., Qi, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+in+Genome+Editing+and+Beyond.">CRISPR/Cas9 in Genome Editing and Beyond.</a> Annual Review of Biochemistry. 85 (1), 227-264 (2016).</li><li>Awata, H., Watanabe, T., Hamanaka, Y., Mito, T., Noji, S., Mizunami, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockout+crickets+for+the+study+of+learning+and+memory:+Dopamine+receptor+Dop1+mediates+aversive+but+not+appetitive+reinforcement+in+crickets.">Knockout crickets for the study of learning and memory: Dopamine receptor Dop1 mediates aversive but not appetitive reinforcement in crickets.</a> Scientific Reports. 5, 15885 (2015).</li><li>Horch, H. W., Liu, J. J., Mito, T., Popadic, A., Watanabe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+in+the+Cricket.">Protocols in the Cricket.</a> The Cricket as a Model Organism: Development, Regeneration, and Behavior. , 327-370 (2017).</li><li>Watanabe, T., Noji, S., Mito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Editing+in+the+Cricket,+Gryllus+bimaculatus.">Genome Editing in the Cricket, Gryllus bimaculatus.</a> Genome Editing in Animals. , 219-233 (2017).</li><li>Kainz, F., Ewen-Campen, B., Akam, M., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch/Delta+signalling+is+not+required+for+segment+generation+in+the+basally+branching+insect+Gryllus+bimaculatus.">Notch/Delta signalling is not required for segment generation in the basally branching insect Gryllus bimaculatus.</a> Development. 138 (22), 5015-5026 (2011).</li><li>Miyawaki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+Wingless/Armadillo+signaling+in+the+posterior+sequential+segmentation+in+the+cricket,+Gryllus+bimaculatus+(Orthoptera),+as+revealed+by+RNAi+analysis.">Involvement of Wingless/Armadillo signaling in the posterior sequential segmentation in the cricket, Gryllus bimaculatus (Orthoptera), as revealed by RNAi analysis.</a> Mechanisms of Development. 121 (2), 119-130 (2004).</li><li>Donoughe, S., Kim, C., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+live-imaging+of+embryos+in+microwell+arrays+using+a+modular+specimen+mounting+system.">High-throughput live-imaging of embryos in microwell arrays using a modular specimen mounting system.</a> Biology Open. 7 (7), bio031260 (2018).</li><li>Donoughe, S., Nakamura, T., Ewen-Campen, B., Green, D. A., Henderson, L., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+signaling+is+required+for+the+generation+of+primordial+germ+cells+in+an+insect.">BMP signaling is required for the generation of primordial germ cells in an insect.</a> Proceeding of the National Academy of Science USA. 111 (11), 4133-4138 (2014).</li><li>Larson, E., Andres, J., Harrison, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+the+male+ejaculate+on+post-mating+prezygotic+barriers+in+field+crickets.">Influence of the male ejaculate on post-mating prezygotic barriers in field crickets.</a> PLOS ONE. 7 (10), e46202 (2012).</li><li>Donoughe, S., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+development+of+the+cricket+Gryllus+bimaculatus.">Embryonic development of the cricket Gryllus bimaculatus.</a> Developmental Biology. , (2016).</li><li>Matsuoka, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+germ+insects+utilize+both+the+ancestral+and+derived+mode+of+Polycomb+group-mediated+epigenetic+silencing+of+Hox+genes.">Short germ insects utilize both the ancestral and derived mode of Polycomb group-mediated epigenetic silencing of Hox genes.</a> Biology Open. 4 (6), 702-709 (2015).</li><li>Rosenberg, M., Lynch, J., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heads+and+tails:+Evolution+of+antero-posterior+patterning+in+insects.">Heads and tails: Evolution of antero-posterior patterning in insects.</a> Biochimica et biophysica acta. 1789 (4), 333-342 (2009).</li><li>Bacon, J., Strausfeld, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonrandom+resolution+of+neuron+arrangements.">Nonrandom resolution of neuron arrangements.</a> Neuroanatomical Techniques: Insect Nervous System. , 357-372 (1980).</li></ol>

The authors have nothing to disclose.

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...

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&#39;s adherence to Krogh&#39;s principle, that for every biological question there is an organism best suited to its solution2,3, 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 piggyBac7,8. 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 lines12,13 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 development14,15. 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 elsewhere12,13, these approaches will ultimately rely on the injection protocol detailed here.

<table>
	<tbody>
		<tr>
			<td>Fluorescent dissecting microscope</td>
			<td>Leica</td>
			<td>M165 FC</td>
			<td>Stereomicroscope with fluorescence</td>
			<td></td>
		</tr>
		<tr>
			<td>External light source for fluorescence</td>
			<td>Leica</td>
			<td>EL 6000</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Narishige</td>
			<td>IM-300</td>
			<td>-Accessories may include Injection Needles Holder, Input Hose (with a hose connector), AC Power Cord, Foot Switch, Silicone Rubber Gasket-</td>
			<td></td>
		</tr>
		<tr>
			<td>mCherry filter cube</td>
			<td>Leica</td>
			<td>M205FA/M165FC</td>
			<td>Filter cube for mCherry or similar red dye will work</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments, Inc.</td>
			<td>M3301R</td>
			<td>Used with Magnetic Stand (Narishige, Type GJ-8)</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>Narishige</td>
			<td>MMO-202ND</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Holder (Needle holder)</td>
			<td>Narishige</td>
			<td>HD-21</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Tubing to connect air source to microinjector</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Egg well stamp</td>
			<td>3D printed</td>
			<td>custom</td>
			<td>3D printed on a Lulzbot Taz 5 using Poly Lactic Acid thermoplastic</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>various</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator or temperature controlled room</td>
			<td>various</td>
			<td></td>
			<td>Temperatures of 23.5-26&#176;C are needed.</td>
			<td></td>
		</tr>
		<tr>
			<td>cricket food</td>
			<td>various</td>
			<td></td>
			<td>cat food or fish flakes are appropriate food.&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>cricket wter</td>
			<td>vairous</td>
			<td></td>
			<td>Water can be held in vials and presented to crickets through cotton balls</td>
			<td></td>
		</tr>
		<tr>
			<td>cricket shelter</td>
			<td>arious</td>
			<td></td>
			<td>Shelter materials can include crumpled paper towels or egg cartons</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillary tubes</td>
			<td>World Precision Instruments, Inc.</td>
			<td>Item no. 1B100F-4</td>
			<td>Kwik-Fil&#8482; Borosilicate Glass Capillaries, 100mm length, 0.58 mm ID, 1.0 mm OD, with filament</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Flaming/Brown</td>
			<td>Model P-97</td>
			<td>Distributed by Sutter Instrument Co.</td>
			<td></td>
		</tr>
		<tr>
			<td>Beveller/Micro grinder</td>
			<td>Narishige</td>
			<td>Model EG-45/EG-400</td>
			<td>EG-400 includes a microscope head</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>CellTreat</td>
			<td>Product code 229693</td>
			<td>90mm diameter</td>
			<td></td>
		</tr>
		<tr>
			<td>Play Sand</td>
			<td>Sandtastik Products Ltd.</td>
			<td>B003U6QLVS</td>
			<td>White play sand</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>American Bioanalytical</td>
			<td>AB000972</td>
			<td>Agarose GPG/LE ultrapure</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg Strainer: Extra Fine Twill Mesh Stainless Steel Conical Strainers</td>
			<td>US Kitchen Supply</td>
			<td>Model SS-C123</td>
			<td>Pore size should be between 0.5 - 1.0 mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco by Life Technologies</td>
			<td>Ref 15070-063</td>
			<td>Pen Strep</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tweezers</td>
			<td>Sipel Electronic SA</td>
			<td>P3C-STD</td>
			<td>Black Static Dissipative, 118mm</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe filters, 25mm diameter, 0.45 &#181;m</td>
			<td>Nalgene</td>
			<td>725-2545</td>
			<td>Use with 1 ml syringe</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe, with Tuberculin Slip Tip</td>
			<td>Becton Dickinson</td>
			<td>309602</td>
			<td>Use with syring filter to filter Injection Buffer , Luer-Lok tip syringes would also work</td>
			<td></td>
		</tr>
		<tr>
			<td>Air tank (optional)</td>
			<td>Midwest Products</td>
			<td>Air Works&#174;</td>
			<td>Portable air tank</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Rhodamine dye</td>
			<td rowspan="2">Thermofisher</td>
			<td rowspan="2">D-1817</td>
			<td rowspan="2">dextran, tetramethylrhodamine 10,000MW,</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL loading tips</td>
			<td>Eppendorf</td>
			<td>Order no. 5242 956.003</td>
			<td>epT.I.P.S. 20uL Microloader</td>
			<td></td>
		</tr>
		<tr>
			<td>Compound microscope</td>
			<td>Zeiss</td>
			<td>Axioskope 2 plus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20X objective</td>
			<td>Ziess</td>
			<td>Plan-Apochromat 20x/0.75 M27</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Leica</td>
			<td>DMC 5400</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite&#160; software</td>
			<td>Leica</td>
			<td>LAS</td>
			<td>Version 4.6.2 used here</td>
			<td></td>
		</tr>
	</tbody>
</table>

injecting gryllus bimaculatus eggs

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.

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&#8211;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...

Watch this Scientific Journal Video about Injecting Gryllus bimaculatus Eggs at JoVE.com

Injecting Gryllus bimaculatus Eggs

Injektion von Gryllus bimaculatus Eiern

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.

Injecting Gryllus bimaculatus Eggs

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been ...

Research

JoVE Journal

Developmental Biology

16.2K Aufrufe.  Colby College. Diese Technik dient als grundlegende Methode für Experimente, die entwickelt wurden, um die embryonale oder Larvenentwicklung des Cricket zu assayen. Der Hauptvorteil dieser Technik ist, dass sie flexibel ist und verschiedene Arten von experimentellen Ansätzen unterstützt, wie z. B. solche, die RNA-Interferenz oder genomische Manipulation nutzen. Demonstriert wird das Verfahren von Hadley Wilson Horch, dem Hauptermittler des Labors.Um dieses Verfahren zu beginnen, nass die Spinnschl...

Serie: Injecting Gryllus bimaculatus Eggs

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L&#8722;1 in different concentrations of ERM at pH 7 and 25 &#176;C in the dark.
We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Salinität abhängigen Toxizität Assay von Silbernanokolloide Verwendung von Medaka-Eier

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

Forschung

Entwicklungsbiologie

In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth

Within the past three decades, red meat and poultry scientists focused on developing strategies and technologies to manipulate muscle development during embryonic and fetal development. This area continues to be an area of focus because muscle fiber number is established during this time and determines the basis for all future growth. In poultry, numerous studies demonstrated in ovo feeding of growth factors, vitamins, or other nutrients improved chick embryonic muscle and intestinal development. Improving in ovo muscle development could benefit the poultry industry by possibly influencing meat yield, growth rate, or myopathy conditions. During the past five years, the Gonzalez Laboratory at the University of Georgia developed a nicotinamide riboside in ovo feeding methodology for broiler-chicken embryos, which altered&#160;muscle development. When injected into a developing embryo&#39;s yolk sac, nicotinamide riboside increased pectoralis major muscle weight and muscle fiber density at hatch. This protocol will demonstrate a methodology to accurately and reproducibly conduct in ovo feeding studies utilizing commercial standard- and high-yielding broiler embryos. These data and methods will allow other research groups to perform in ovo feeding studies with much success and reproducibility.

In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth

A robust methodology has been developed for conducting in ovo feeding research trials utilizing unincubated commercial broiler eggs to test the ability of natural and synthetic compounds, in this case, nicotinamide riboside, to influence muscle development and growth.

In Ovo Fütterung von kommerziellen Masthähncheneiern: eine genaue und reproduzierbare Methode, um die Muskelentwicklung und das Muskelwachstum zu beeinflussen

Within the past three decades, red meat and poultry scientists focused on developing strategies and technologies to manipulate muscle development during ...

Untersuchung der Entwicklung mit optogenetischer Signalmanipulation im Zebrafisch

Optogenetic Signaling Activation in Zebrafish Embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic&#160;tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

Autor im Rampenlicht: Manipulation der Signalübertragung in Zebrafischembryonen, um Entscheidungen über das Schicksal von Zellen zu entschlüsseln 

Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo.

Optogenetische Signalaktivierung in Zebrafischembryonen

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...

Verbesserung der Proteinvisualisierung mit Antikörper-vermittelter Live-Bildgebung

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

Autor im Rampenlicht: Enthüllung der Dynamik mechanischer und biochemischer Signale in der Morphogeneseforschung an Tieren 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

Visualisierung von Proteinen mit geringer Häufigkeit und posttranslationalen Modifikationen in lebenden Drosophila-Embryonen mittels Fluoreszenz-Antikörper-Injektion

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...