Name: injecting gryllus bimaculatus eggs
Uploaded: 2019-08-22T13:00:00
Description: Watch this Scientific Journal Video about Injecting Gryllus bimaculatus Eggs at JoVE.com

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 microma...

<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 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

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

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 ...

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

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 ...

Investigating Development with Optogenetic Signaling Manipulation in Zebrafish

Optogenetic Signaling Activation in Zebrafish Embryos

Manipulating Signaling in Zebrafish Embryos to Decode Cell Fate Decisions (Video) | JoVE

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

Enhancing Protein Visualization with Antibody-Mediated Live Imaging

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

Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research (Video) | JoVE

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