Name: maintaining laboratory cultures of gryllus bimaculatus, a versatile orthopteran model for insect agriculture and invertebrate physiology
Uploaded: 2022-06-08T12:30:00
Description: Watch this Scientific Journal Video about Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology at JoVE.com

Funding for this project was made possible through University of Wisconsin-Madison internal grants. Sincerest thanks to Kevin Bachhuber of Bachhuber Consulting Inc. for access to his unpublished guide for commercial cricket rearing and to Michael Bartlett Smith for his assistance in refining and troubleshooting these methods.

1. Preparing the oviposition substrate
NOTE: Coconut coir is an ideal oviposition substrate for G. bimaculatus. For detailed methods on how to separate coir from compressed coir brick and a note on respiratory safety, see Supplemental Materials step 1.1.
<ol>
	<li>Wash hands with soap and water.</li>
	<li>Tare a clean container on a balance and weigh a mass of dry coconut coir approximately the size of a human fist.</li>
	<li>Place coir into a seala...

<ol>
	<li>Hales, K. G., Korey, C. A., Larracuente, A. M., Roberts, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+on+the+fly:+a+primer+on+the+Drosophila+model+system.">Genetics on the fly: a primer on the Drosophila model system.</a> Genetics. 201 (3), 815-842 (2015).</li><li>Merkel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+temperature+and+food+quality+on+the+larval+development+of+Gryllus+bimaculatus+(Orthoptera,+Gryllidae).">The effects of temperature and food quality on the larval development of Gryllus bimaculatus (Orthoptera, Gryllidae).</a> Oecologia. 30 (2), 129-140 (1977).</li><li>Bateman, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mate+preference+for+novel+partners+in+the+cricket+Gryllus+bimaculatus.">Mate preference for novel partners in the cricket Gryllus bimaculatus.</a> Ecological Entomology. 23 (4), 473-475 (1998).</li><li>Mito, T., 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=The+two-spotted+cricket+Gryllus+bimaculatus:+An+emerging+model+for+developmental+and+regeneration+studies.">The two-spotted cricket Gryllus bimaculatus: An emerging model for developmental and regeneration studies.</a> Cold Spring Harbor Protocols. 2008, (2008).</li><li>Ylla, G., 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=Cricket+genomes:+the+genomes+of+future+food.">Cricket genomes: the genomes of future food.</a> BioRxiv. , (2020).</li><li>Halloran, A., Roos, N., Hanboonsong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cricket+farming+as+a+livelihood+strategy+in+Thailand.">Cricket farming as a livelihood strategy in Thailand.</a> Geographical Journal. 183 (1), 112-124 (2017).</li><li>Wade, M., Hoelle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+edible+insect+industrialization:+scales+of+production+and+implications+for+sustainability.">A review of edible insect industrialization: scales of production and implications for sustainability.</a> Environmental Research Letters. 15, 123013 (2020).</li><li>. Studies on the influence of different diets and rearing conditions on the development and growth of the two-spotted cricket Gryllus bimaculatus de Greer Available from: <a target="_blank" href="https://epub.uni-bayreuth.de/310/1/Diss.pdf">https://epub.uni-bayreuth.de/310/1/Diss.pdf</a> (2011)</li><li>Ngonga, C. A., Gor, C. O., Okuto, E. A., Ayieko, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+performance+of+Acheta+domesticus+and+Gryllus+bimaculatus+production+reared+under+improvised+cage+system+for+increased+returns+and+food+security.">Growth performance of Acheta domesticus and Gryllus bimaculatus production reared under improvised cage system for increased returns and food security.</a> Journal of Insects as Food and Feed. 7, 301-310 (2021).</li><li>Behrens, W., Hoffmann, K. -. H., Kempa, S., G&#228;&#223;ler, S., Merkel-Wallner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+diurnal+thermoperiods+and+quickly+oscillating+temperatures+on+the+development+and+reproduction+of+crickets,+Gryllus+bimaculatus.">Effects of diurnal thermoperiods and quickly oscillating temperatures on the development and reproduction of crickets, Gryllus bimaculatus.</a> Oecologia. 59 (2-3), 279-287 (1983).</li><li>Collavo, A., Paoletti, M. G., 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=Housecricket+smallscale+farming.">Housecricket smallscale farming.</a> Ecological implications of minilivestock. Potential of insects, rodents, frogs and snails. , (2005).</li><li>Simmons, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competition+between+larvae+of+the+field+cricket,+Gryllus+bimaculatus+(Orthoptera:+Gryllidae)+and+its+effects+on+some+life-history+components+of+fitness.">Competition between larvae of the field cricket, Gryllus bimaculatus (Orthoptera: Gryllidae) and its effects on some life-history components of fitness.</a> Journal of Animal Ecology. 56, 1015-1027 (1987).</li><li>Sorjonen, J. M., 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=The+plant-based+by-product+diets+for+the+mass-rearing+of+Acheta+domesticus+and+Gryllus+bimaculatus.">The plant-based by-product diets for the mass-rearing of Acheta domesticus and Gryllus bimaculatus.</a> PLOS ONE. 14 (6), 0218830 (2019).</li><li>Maciel-Vergara, G., Jensen, A. B., Lecocq, A., Eilenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diseases+in+edible+insect+rearing+systems.">Diseases in edible insect rearing systems.</a> Journal of Insects as Food and Feed. 7 (5), 1-18 (2021).</li><li>Alexander, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggressiveness,+territoriality,+and+sexual+behavior+in+field+crickets+(Orthoptera:+Gryllidae).">Aggressiveness, territoriality, and sexual behavior in field crickets (Orthoptera: Gryllidae).</a> Behaviour. , 130-223 (1961).</li><li>Pet food, fish bait, and animal feed. USDA APHIS Available from: <a target="_blank" href="https://www.aphis.usda.gov/aphis/ourfocus/planhealth/import-information/permits/plant-pests/sa_animalfeed/ct_petfood_fishbait_animalfeed">https://www.aphis.usda.gov/aphis/ourfocus/planhealth/import-information/permits/plant-pests/sa_animalfeed/ct_petfood_fishbait_animalfeed</a> (2022)</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. 411 (1), 140-156 (2016).</li><li>Dobermann, D., Michaelson, L., Field, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+an+initial+high-quality+feeding+regime+on+the+survival+of+Gryllus+bimaculatus+(black+cricket)+on+bio-waste.">The effect of an initial high-quality feeding regime on the survival of Gryllus bimaculatus (black cricket) on bio-waste.</a> Journal of Insects as Food and Feed. 5 (2), 1-8 (2018).</li><li>Lundy, M. E., Parrella, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crickets+are+not+a+free+lunch:+Protein+capture+from+scalable+organic+side-streams+via+high-density+populations+of+Acheta+domesticus.">Crickets are not a free lunch: Protein capture from scalable organic side-streams via high-density populations of Acheta domesticus.</a> PLOS ONE. 10, 0118785 (2015).</li><li>Mazaya, G., Karseno, K., Yanto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+and+phytochemical+activity+of+coconut+shell+extracts.">Antimicrobial and phytochemical activity of coconut shell extracts.</a> Turkish Journal of Agriculture - Food Science and Technology. 8 (5), 1090-1097 (2020).</li></ol>

The simplicity of this approach to cricket rearing can benefit a range of research areas and represents a generic template for successful cricket husbandry, easily adaptable to a variety of experimental needs. Compared to several other studies of G. bimaculatus, the individual body adult size is smaller and maturation is slower14, which we attribute to sub-optimal rearing temperature imposed on us by circumstance. The methods described above have been used and refined over the course of 2...

As typified by the iconic insect, the fruit fly Drosophila melanogaster, the use of insects as laboratory model organisms provides distinct advantages for studies in genetics, toxicology, and physiology1. The small size of insects reduces the space needed for cultures and the amount of feed and consumable materials required. Many insects&#160;reproduce quickly making them uniquely suited to the creation of specialized genetic lines and studies requiring the evaluation of multiple successive generations.
Many studies focus on holometabolous insects such as Drosophila, which exhibit complete metamorphosis and pupation. However, other models are available, including Gryllus bimaculatus (De Geer),&#160;the two-spotted field cricket. G. bimaculatus is a paurometabolous insect that undergoes between 7 and 11 nymphal instars before reaching sexual maturity2. This cricket displays a wide range of behaviors related to sexual selection, including stridulation, territorial displays, and mate-guarding3. Immature crickets are unlike the larvae of holometabolous insect species in that they, similar to many paurometabolous juveniles, are able to regenerate lost and damaged limbs during ecdysis4. Additionally, the fully sequenced genome of G. bimaculatus was published in 20215. These characteristics make these crickets appealing as a target for basic research.
Two-spotted field crickets are widely reared for human food and animal feed. The scale of these operations is often much larger than for laboratory research6,7. Despite the difference in scale, the challenges faced by researchers overlap greatly with those encountered by commercial cricket farmers. These considerations converge in the context of lab-based research aiming to improve edible insect production. As the edible insect industry continues to evolve and grow, optimizing feed inputs and myriad other aspects of production is a primary goal8. Laboratory studies demonstrating measured improvements in rearing efficiency, survivorship, or generation time in these crickets have the potential to help increase the profitability of cricket farming operations long-term.
Standardized rearing protocols enable closer comparison between studies investigating rearing optimization. To-date, few in-depth protocols for rearing G. bimaculatus in the laboratory have been published. An ideal protocol would reflect conditions encountered in real-world cricket farming operations, while maintaining the strictly controlled conditions necessary to accurately measure changes in growth performance arising from experimental treatments and highlighting risk mitigation strategies. The methods described in this paper were developed based on published protocols, techniques, and apparatus used to rear a variety of cricket species at a broad range of laboratory and commercial production scales2,9,10,11,12. These methods are also informed by several non-peer reviewed sources, including unpublished technical bulletins and personal communication with commercial cricket farmers in North America. This protocol was developed with the intention of facilitating the establishment of laboratory cultures of G. bimaculatus specifically for use in trials related to insect agriculture.

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			<td>31-qt (29.3 L) Snap-lid tote bin with lid</td>
			<td>HOMZ</td>
			<td>3430CLBL</td>
			<td>Used to house breeding stock</td>
		</tr>
		<tr>
			<td>3-tier/12-tray Grow Light Stand</td>
			<td>Fischer Scientific</td>
			<td>NC1938548</td>
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			<td>50-gal (189.27L) tote bin with lid</td>
			<td>Sterilite</td>
			<td>#14796603</td>
			<td>Used as secondary containment when handling crickets</td>
		</tr>
		<tr>
			<td>50 mL polypropylene graduated cylinder</td>
			<td>Fischer Scientific</td>
			<td>S95171</td>
			<td></td>
		</tr>
		<tr>
			<td>7.5-qt (7.1 L) snap-lid tote bin with lid</td>
			<td>HOMZ</td>
			<td>3410CLBL</td>
			<td>Used to house exprimental stock</td>
		</tr>
		<tr>
			<td>Accuris 500 g x 0.01 g Balance</td>
			<td>Manufactured by Accuris, a subsidieary of Benchmark Scientific</td>
			<td>W3300-500</td>
			<td>Purchased from Dot Scientific through University of Wisconsin system purchasing service &#34;ShopUW+&#34;</td>
		</tr>
		<tr>
			<td>Ace Premier 1 Inch Flat Chip Brush</td>
			<td>Ace Hardware&#160;</td>
			<td>#1803261</td>
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			<td>Bel-Art SP Scienceware deionized water wash bottle</td>
			<td>Fischer Scientific</td>
			<td>03-421-160&#160;</td>
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			<td>Bright aluminum window screen&#160;</td>
			<td>Phifer</td>
			<td>UNSPSC# 11162108</td>
			<td>Mesh size 18 x 16&#34;</td>
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			<td>Clear Disposable Plastic Portion Cups 5.5 oz w/ lids</td>
			<td>Wal-Mart</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>Diablo 4-4/8&#34; x 13 TPI Ultra Fine Finish Bi-Metal Jigsaw Blade</td>
			<td>Home Depot</td>
			<td>#313114935</td>
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			<td>Egg Filler Flats-Paper, 12 x 12&#34;</td>
			<td>Uline</td>
			<td>S-5189</td>
			<td></td>
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			<td>Fisherbrand Petri Dishes with Clear Lid 100 x 15mm</td>
			<td>Fischer Scientific</td>
			<td>FB0875714</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Petri Dishes with Clear Lid 60 x 15mm</td>
			<td>Fischer Scientific</td>
			<td>FB0875713A</td>
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			<td>Home Depot</td>
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			<td>Infinity Bond</td>
			<td>Infinity IM-Tough-Guy-12</td>
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			<td>Mazuri Cricket Diet</td>
			<td>Land O&#39; Lakes International</td>
			<td>SKU#&#160; 3002219-105</td>
			<td></td>
		</tr>
		<tr>
			<td>Stanley TimeIt Twin 2-outlet Grounded Mechanical 24 Hour Timer</td>
			<td>Wal-Mart</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Vermont Organics Reclamation Soil 11 lb Coir Block</td>
			<td>Home Depot</td>
			<td>#300679904</td>
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	</tbody>
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maintaining laboratory cultures of gryllus bimaculatus, a versatile orthopteran model for insect agriculture and invertebrate physiology

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human food. Outside its native range, culturing G. bimaculatus is feasible due to its dietary plasticity, rapid reproductive cycle, lack of diapause requirement, tolerance for high-density rearing, and robustness against pathogens. Thus, G. bimaculatus can be&#160;a versatile model for studies of insect physiology, behavior, embryology, or genetics.
Cultural parameters, such as stocking density, within-cage refugia, photoperiod, temperature, relative humidity, and diet, all impact cricket growth, behavior, and gene expression and should be standardized. In the burgeoning literature on farming insects for human consumption, these crickets are frequently employed to evaluate candidate feed admixtures derived from crop residues, food-processing byproducts, and other low-cost waste streams.
To support ongoing experiments evaluating G. bimaculatus growth performance and nutritional quality in response to variable feed substrates, a comprehensive set of standard protocols for breeding, upkeep, handling, measurement, and euthanasia in the laboratory was developed and is presented here. An industry-standard cricket feed has proven nutritionally adequate and functionally appropriate for the long-term maintenance of cricket breeding stocks, as well as for use as an experimental control feed. Rearing these crickets at a density of 0.005 crickets/cm3 in screen-topped 29.3 L polyethylene cages at an average temperature of 27 &#176;C on a 12 light (L)/12 dark (D) photoperiod, with moistened coconut coir serving both as hydration source and oviposition medium has successfully sustained healthy crickets over a 2-year span. Following these methods, crickets in a controlled experiment yielded an average mass of 0.724 g 0.190 g at harvest, with 89% survivorship and 68.2% sexual maturation between stocking (22 days) and harvest (65 days).

Data demonstrating successful cricket rearing from hatching to 65 days old were collected during a September 2021 feed trial. Crickets were grown from eggs following steps 1.1.1-2.6.1 of these protocols, and six replicate cages were stocked with 24 random 22-day-old (third instar) crickets following step 2.7 above. Crickets were then reared in ambient room conditions; however, due to a malfunctioning facility air handling unit, the average room temperature was 25 &#177; 1 &#176;C at 20% relative humidity rather than the ...

Watch this Scientific Journal Video about Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology at JoVE.com

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

This paper outlines basic methods to standardize important factors such as density, feed availability, hydration source, and environmental controls for the long-term rearing of laboratory cultures of the edible cricket, Gryllus bimaculatus.

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human ...

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