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

These techniques depict practical and proven methods for rearing Gryllus bimaculatus, a key mini livestock species farmed for animal feed and human food and increasingly important alternative model organism. These laboratory methods for the culture of Gryllus bimaculatus are derived from techniques common throughout the North American cricket farming industry and represent a laboratory-scale facsimile of cricket farming operations. To begin, tare a clean container on a balance, and weigh a mass of dry coconut coir approximately the size of a human fist.

Then, place coir into a sealable, clean container, which can accommodate expansion up to six times the original volume. With clean hands, gently break up clumps of coir from the piece removed from the larger block. Next, using a 250-milliliter graduated cylinder, measure the correct volume of deionized water to achieve a five-to-one ratio by mass of five parts water to one part dry coir, and add the measured deionized water slowly, evenly hydrating all coir particles.

Afterward, manually macerate clumps to ensure even hydration, and re-tare the container in which the coir was previously weighed. Weigh out 75 grams of wetted coir, and transfer it into a 100-millimeter-by-15-millimeter Petri dish using a clean, plastic spoon to ensure that the coir is evenly spread around the bottom of the dish and that there are no clumps. Then, label the side of the Petri dish with lab tape with a labeled denoting natal colony and date of the egg collection event.

Measure an additional 45 milliliters of deionized water into a graduated cylinder, and add water evenly over the surface of the packed coir in the Petri dish to ensure even hydration. Once the Petri dish has been packed, seal the remaining wetted coir in an airtight vessel for storage at minus 20 degrees Celsius. Place the hydrated oviposition substrate into cages containing the desired parental stocks of crickets as far from the feed as possible, and document the date and time.

Then, place a small, autoclavable trash receptacle on the work surface. Next, place a clean, empty, 29.3 liters plastic cage on the bench next to the trash receptacle. Place another 29.3 liters cage containing the parent cricket stocks and oviposition substrate on the opposite side of the trash receptacle from the empty cage.

After 24 hours, remove the oviposition substrate from the parent cricket cage, and locate it over autoclavable waste vessel. Inspect the top of the oviposition substrate for any particles of frass or feed that the crickets may have kicked onto the surface of the coir. Next, remove coir contaminants into the waste vessel with a clean Scoopula or plastic spoon, and place the plastic spoon into the waste receptacle.

Then, place the cleaned oviposition substrate in the clean, 29.3-liter cage. Afterward, place the cage in an incubator set to 27 degrees Celsius at 60%relative humidity on a 12-hour dark, 12-hour light photoperiod. Return the cage containing the breeding stock to the original location, and clear all items from the work surface.

Then, place the waste receptacle into an in-facility freezer dedicated to the storage of items potentially contaminated with cricket eggs. Next, sanitize the work surface with 10%bleach solution, and let it sit for 60 seconds. Wipe the work surface dry with a clean paper towel.

After opening the freezer, dispose of the paper towel in the waste receptacle. Select two unused 30.8-centimeters-by-30.8-centimeters commercial egg carton flats. And with a utility knife or strong shears, cut these into six separate 10.1-centimeter-wide strips of equal size.

Then, brush off the cut edges with hands to remove dangling particles of cardboard. Next, place the six individual 10.1-centimeters-by-30.8-centimeters pieces of carton vertically into the bottom of the cage, with the longer axis of the carton spanning the narrower horizontal axis of a 29.3-liter cage. Place a seventh piece of carton flat across the top of the six upright pieces.

After selecting three pieces of rough, brown paper towel approximately 25 centimeters by 25 centimeters, fold each in half. Then, place two such that they cover the top of the proximal side of the carton structure, and place one over the carton stack on the distal side. At day 11 post-oviposition, move the oviposition substrate into the proximal right-hand corner of the cage.

When a hatch is observed, mist the paper towels placed over the top of the cartons in, until they are wetted, but not actively shedding water. Then, thoroughly wipe both sides of a 100-millimeter Petri lid with 70%ethanol, and allow it to dry. Next, scoop 50 grams of the feed into a 60-watt, single-serving blender, and mill at 10, 000 rpm for one minute.

After measuring one gram of the feed, shake it onto the Petri dish lid in the cage. Using the clean end of a spoon or Scoopula, spread the feed out as uniformly as possible over the bottom of the dish. After, replace the feed every two days.

Discard the old feed in the autoclavable waste container. Monitor for mold growth on feed. And if the feed begins to appear white or greenish, discard the Petri dish and feed immediately.

At 14 days post-oviposition, use a 2.54-centimeter paintbrush to remove crickets clinging to the natal coir substrate by brushing all crickets from the coir surface and sides of the Petri dish into the cage. Next, place the removed oviposition substrate into the autoclavable waste receptacle, and store it in the freezer until autoclaving. Then, replace the natal substrate with a fresh coir dish for hydration.

Using a deionized water wash bottle, add water until the surface of the coir glistens, but is not pooling. Crickets were stocked from a population with mean mass of 21 milligrams. At the end of the experiment, the mean mass of all combined juvenile and adult crickets was 0.724 grams.

At 65 days of age, when the experiment ended, 30 adult males were present, and the mean mass was 0.721 grams, whereas for the 58 adult females, the mean mass was 0.841 grams. Survivorship between stocking and harvest was 89%and was measured weekly by total counts of all individual crickets in all cages. At day 65, approximately 68%of all crickets had reached the adult instar.

Avoid the formation of water droplets on enclosure bottom when watering or misting. Placing paper towel over refugia allows for a humidity gradient and absorbs water droplets from misting. This procedure serves as a template for researchers in many life sciences fields to rear Gryllus bimaculatus as a model organism.

To date, few formal protocols for rearing Gryllus bimaculatus or any other paurometabolous model organism have been published. Codifying and standardizing rearing methods for insects used in insect agriculture will better enable comparison of results between research groups.