Exploring Life History Choices: Using Temperature and Substrate Type as Interacting Factors for Blowfly Larval and Female Preferences

Blow flies are a diverse group of flies that can be obligate or facultative parasites, as well as sapro-necrophagous. Obligate parasitic species feed exclusively on living tissues and cause myiasis in vertebrates, while sapro-necrophagous species feed on decaying organic matter. Facultative parasites can feed on both living or decaying material and live as parasites or not.

Although the larval stage is the most problematic one, the female plays a decisive role as they choose where to oviposit and therefore, largely control where the larvae will feed and develop. The overall goal of the methods presented here are to assess both larval food substrate preference and female oviposition site choice in blow flies. Larval feeding preference.

Step one, preparation of the Petri dishes with agar 2%Prepare Petri dishes with 2%agar. In order to do that, add six grams of bacteriological agar to 300 milliliters of water, and melt this mix in a microwave. Then divide the volume into four Petri dishes.

Prepare the number of Petri dishes with agar according to the number of replicates desire. After the agar is solidified, punch four holes in the agar using the cutting petter provided in our paper in a 50-milliliter conical tube with a three-centimeter diameter, two holes on each side of the Petri dish. Step two, preparation of substrates.

To prepare the fresh meat with blood, use 12 milliliters of diluted bovine blood which is half blood with an anticoagulant solution and half filtered water. Then add this solution to 200 grams of fresh bovine ground beef and stir it well. Remember to use different graduate cylinders and spoons for each type of meat to avoid other contamination between substrates.

To prepare the rotten substrate, add 12 milliliters of filtered water to 200 grams of rotten meat and stir it just like it was done with the fresh meat. Prior to the start of the experiment, the meat is left to rot for five days at 25 degrees Celsius in an incubator. After preparing the meat substrate, fill the holes in the agar with fresh and rot meat on each side.

Remember to randomize the position of the meat in the Petri dishes as we are showing here. Step three, experimental setup. In a room at 25 degrees Celsius, set the heating pad right below a light source to evenly illuminate the preference assay in a wide behavior bias towards or against the light.

Then use paperboard around the heating pad as a support to keep all at the same level. Place a black cardboard over all the set, the heating pad, plus support paperboard, to keep the same color pattern above all substrates. Place six Petri dishes with agar and meat substrate over the black cardboard with two substrates, one of each type on the heating pad and the other two outside the heating pad surface.

Doing this, we will allow each one of the Petri dish to have four substrates, fresh and cold, rotten and cold, fresh and hot, and finally, rotten and hot. Let the substrate heat for about 10 minutes. Step four, larval test.

Check the temperature of the substrate with an infrared thermometer. The colder side should be at around 25 degrees Celsius and the hotter at around 33 degrees Celsius. After reaching the desired temperature, place five third instar larvae in the center of each Petri dish using a tweezer.

Then cover the Petri dish with the lid, and let the choice experiment run for exactly 10 minutes. During the experiment, the larvae will crawl around the Petri dish and choose among the four options. If any larvae escape, take it using a tweezer, and place it back to the center of the Petri dish.

After 10 minutes, get all the experiments Petri dishes out of the heating pad and place them on a different surface. Then count how many larvae there are in each substrate. We have observed that colorful larvae stay in their chosen substrate.

Female oviposition site preference. Step one, experimental setup. The task can be done on a regular shelf that was previously covered up with black cardboards.

These cardboards, just like in a larval assay, are needed, so no visual cues are available for the adult females. The shelf also needs to be lined evenly with LED strips. In a room at 25 degrees Celsius, place the heating pad in the center of the shelf.

Use paperboard around the heating pad as a support to keep all at the same level. Place a black cardboard over all the set, a heating pad, plus support paperboard to keep the same colored pattern above all substrates. Position the heating pads under two arms of the same cross in between two different crosses.

This will allow for better use of the space in the shelf. Turn on the LED lights and the heating pad prior to the start of the experiment. Remember to always clean up the cross-shaped glasses with 70%ethanol to avoid odor contamination between tests.

Step two, preparation of substrates. Prepare the plastic Petri dishes with five grams of fresh or rotten meat. The substrate is the same as the larval test, fresh and five-day-old rotten.

A total of four Petri dishes are required for each cross, two with fresh meat and two with rotten meat. For the fresh meat, add one millimeter of diluted bovine blood which is half of blood with anticoagulant and half of water. For the rotten meat, add one millimeter of filtered water.

Remember to stir both substrates well and to use different spoons to avoid odor cross-contamination between rotten and fresh meat. Check if the alcohol has completely evaporated on the crosses. Then place four Petri dishes with meat at the extremity of the crosses'arms, one of each type of meat on the heating pad, and the other two outside the heating pad surface.

This will allow each one of the crosses to have four substrates, fresh and cold, rotten and cold, fresh and hot, and finally, rotten and hot. Close the opening of the crosses and let the substrates heat for about 10 minutes. Step three, female test.

Collect grab a females in the fly cage. They're characterized by having an enlarged and whitish yellow abdomen. Separate one female for each cross in individual tubes.

Check the temperature of the substrates with an infrared thermometer. The colder side should be at around 25 degrees Celsius and the hotter at around 33 degrees Celsius. Place one tube with a grabbed female right in the center opening of each cross.

After the female enter the cross, close the opening with the lid. After placing all the females in each cross, close the open part of the shelf with the black cardboard to shield against external light sources. Let the experiment run for exactly four hours.

After this time is finished, remove the female from the crosses using a tube. Then check if there were any other position on the substrate. Identify the lid of each Petri dish with the name of the substrate.

Use 70%ethanol to clean up the crosses from any odor from the test. Step four, egg count. The Petri dishes can be frozen prior to counting.

In this case, thaw the Petri dishes. Then, count the number of eggs in each substrate using a stereomicroscope. Data analysis and statistics.

Step one, preference index calculation. For each replicate of the behavioral tests, a preference index is calculated separately for the type of substrate and for the temperature. The preference index for substrate type is represented by the number of larvae or eggs on fresh meat substrates divided by the total number of larvae or eggs on all substrates.

The preference index for temperature is represented by the number of larvae or eggs on hot substrates divided by the total number of larvae or eggs on all substrates. Considering both index preferences, values close to one reflect the preference for fresh or hot substrates. And values close to zero mean the preference for rotten or cold substrates.

Step two, comparison of the observed preference to random choice. We can visualize the preference index for temperature on the Y axis and preference index for the meat on the X axis. To determine if the larvae and, or females display a preference for a condition, we compared our results to a random choice using simulated data.

Our larvae results significantly deferred from all simulated datasets in terms of temperature and meat preference. Graphically, it is evident that larvae exhibited a strong preference for the rotten and cold substrate. We found that the female preference for a meat type was significantly different from a random choice in about 30%of the comparisons.

For the temperature, around 70%of our comparisons resulted in a significant preference. Both protocols offer the possibility to test the preference of larvae and females for two interacting factors, type of meat and temperature. The tests are straightforward, and can be adapted to test the preference of other species of the same size and or other conditions.

We know that animal behavior is quite variable and influenced by several environmental factors, so special care should be taken to avoid unequal lighting, lingering smells, and other contamination of substrate through shared utensils. Thanks for watching.