We acknowledge Patr&#237;cia J. Thyssen, Gabriela S. Zampim and Lucas de Almeida Carvalho for providing the L. cuprina colony and for their assistance in setting up the experiment. We would also like to thank Rafael Barros de Oliveira for filming and editing the video. This research was supported by the Developing Nation Research Grant from Animal Behavior Society to V.A.S.C. and by a FAPESP Dimensions US-Biota-S&#227;o Paulo grant to T.T.T. (20/05636-4). S.T. and D.L.F. were supported by a FAPESP (19/07285-7 postdoctoral grant and 21/10022-8 PhD scholarship, respectively). V.A.S.C. and A.V.R. were supported by CNPq PhD scholarships (141391/2019-7, 140056/2019-0, respectively). T.T.T. was supported by CNPq (310906/2022-9).

Fly samples were obtained using traps and not on infested animals. A SISBIO license (67867-1) was issued to collect and keep flies of the Calliphoridae family in captivity in laboratory conditions. Insect samples are exempt from ethical evaluation in research in Brazil. Bovine meat and blood were obtained commercially, and no ethical clearance was required.
1. Larval feeding preference
<ol>
	<li>Preparation of the Petri dishes containing 2% agar
	<ol>
		<li>Prepare four Petri dishes with 2% agar. To do so, add 6 g of bacteriological agar to 300 mL of water and melt this mix in a microwave. Then, divide the volume evenly into four glass Petri dishes (150 x 20 mm), using around 70 mL in each dish. 
		NOTE: Prepare Petri dishes equal to the number of experimental replicates desired. In this study, 36 replicates were used.</li>
		<li>Once the agar has solidified, use a 50 mL conical tube (3 cm diameter) to punch four holes in the agar, two on each side of the Petri dish, following the cutting pattern provided (Figure 1). 
		NOTE: This setup is similar to previously described protocols by Fouche et al. (2021)40 and Boulay et al. (2016)42.</li>
	</ol>
	</li>
	<li>Preparation of substrates
	<ol>
		<li>To prepare the fresh meat with blood, add 12 mL of diluted bovine blood to 200 g of fresh bovine ground meat. Mix thoroughly. Be sure to use different graduated cylinders and spoons for each type of meat to avoid cross-contamination between substrates. 
		NOTE: The diluted blood is made up of 50% pure blood mixed with an anticoagulant (3.8% sodium citrate) and 50% filtered water.</li>
		<li>To prepare the rotten substrate, add 12 mL of filtered water to 200 g of 5-day-old rotten bovine ground meat and mix it well. 
		NOTE: Rotten meat was obtained by incubating fresh ground meat for five days at 25 &#176;C in non-hermetic plastic pots (mix of aerobic and non-aerobic decomposition). Each pot contained 200 g of fresh ground meat. It was then frozen until use.</li>
		<li>Fill two holes in each Petri dish with the fresh meat and blood mixture and the remaining two holes with the rotten meat and water mixture. 
		NOTE: To avoid position biases, vary the placement of the different meat types in the Petri dishes. For example, some Petri dishes should have the same type of meat facing each other, while in other dishes, the meat type should be crossed, as shown in Figure 2.</li>
	</ol>
	</li>
	<li>Experimental setup
	<ol>
		<li>At a room temperature (RT) of 25 &#176;C, position the heating pad directly below a light source to evenly illuminate the experimental area and avoid any behavior bias towards or against the light. Place paperboard pads around the heating pad to ensure that the experimental setup remains leveled. 
		NOTE: The light source used was white low heating emitting light, such as a neon tube. The heating pad was positioned on a table right below the ceiling light bulbs (Figure 3).</li>
		<li>Cover the heating pad and leveling paperboard pads with black cardboard and switch the heating pad on. 
		NOTE: The black cardboard cover should be used to avoid visual cues that may bias the behavior assay.</li>
		<li>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 off the surface of the heating pad (Figure 2). Let the substrates heat for approximately 10 min. 
		NOTE: Condensation may form on the lid of the Petri dishes.</li>
	</ol>
	</li>
	<li>Larval test
	<ol>
		<li>Check the temperature of the substrates (cold side: 25 &#177; 2 &#176;C; hot side: 33 &#177; 2 &#176;C) with an infrared thermometer. 
		NOTE: The heating pad stays on during the whole length of the experiment. Temperature measurements were made at the beginning and at the end of the experiment. Although the temperature did fluctuate by &#177; 2 &#176;C, there was still at least an 8 &#176;C temperature difference between the hot and cold conditions.</li>
		<li>After reaching the desired temperature, place five third-instar larvae in the center of each Petri dish using tweezers (Figure 2) and cover the Petri dishes with the lids. Let the choice experiment run for 10 min. 
		NOTE: Some larvae may crawl around the edges and on the lid of the Petri dishes. If any larva escapes, use a tweezer to place it back to the center of the Petri dish.</li>
		<li>After 10 min, get all the Petri dishes off of the heating pad and place them on a different surface to avoid continuing heating the substrates. Then, count the number of larvae in each substrate, as well as those that did not choose any substrate. 
		&#8203;NOTE: Lucilia cuprina larvae stay in their chosen substrate, as observed in this experiment.</li>
	</ol>
	</li>
</ol>
2. Female oviposition site preference
<ol>
	<li>Experimental setup
	<ol>
		<li>Use a regular shelf previously covered up with black cardboard and evenly illuminated with white LED light strips. 
		NOTE: The black cardboard covers should be used to avoid visual cues that may bias the behavior assay. The white LED strips are secured lengthwise in the middle of the shelf right above the experiment. The shelves used in the setup were placed 45 cm apart.</li>
		<li>At RT (25 &#176;C), place a heating pad in the center of the shelf. Use paperboard pads around the heating pad as a support to ensure that the experimental setup is leveled.</li>
		<li>Cover the heating pad and leveling paperboard pads with a black cardboard to keep the same visual pattern below all the substrates.</li>
		<li>Place two cross-shaped-glass containers on one shelf, each should have two arms over the black cardboard and heating pad. Switch on the white LED light strips and the heating pads prior to the start of the experiment.</li>
		<li>Use 70% ethanol to clean the crosses (inside the cross and the lid) to avoid odor contamination.</li>
	</ol>
	</li>
	<li>Preparation of substrates
	<ol>
		<li>Prepare four Petri dishes (60 mm x 15 mm) per cross with 5 g of 5-day-old rotten or fresh meat (two of each type of substrate). 
		NOTE: Prepare Petri dishes equal to the number of experimental replicates desired times four. In this study 30 replicates were used, totaling 120 prepared Petri dishes.</li>
		<li>Add 1 mL of diluted bovine blood (50% pure blood with anticoagulant and 50% filtered water) onto the fresh meat and 1 mL of filtered water onto the rotten meat. Thoroughly mix the meat (fresh or rotten) with the liquid (blood or water) using a different spoon for each type of meat. 
		NOTE: The preparation of the meat for the female assay is very similar to the larval assay, although the quantities are different because the female test will be running for longer than the larval test. Remember to use different pipette tips and spoons for each type of meat to avoid any odor cross-contamination between substrates.</li>
		<li>Check if the alcohol has completely evaporated from the crosses. Then, place four Petri dishes (one of each type of meat on the heating pad and the other two off the heating pad&#39;s surface) at the extremity of each arm of the cross (Figure 4). Close the crosses with their lids and allow the substrates to heat for approximately 10 min. 
		NOTE: In addition, to avoid position biases, vary the placement of the different meat types in the crosses. For example, some crosses should have the same type of meat on adjacent arms, while in other crosses, the same meat type should be across from one another, as shown in Figure 4.</li>
	</ol>
	</li>
	<li>Female test
	<ol>
		<li>Collect gravid females in the fly cage and isolate them into individual tubes. 
		NOTE: Gravid females are characterized by having an enlarged and whitish-yellow abdomen in contrast to non-gravid females (Figure 5). Gravid females were collected between 10 and 16 days after emergence for the experiments.</li>
		<li>Check the temperature of the substrates in the crosses (cold side: 25 &#177; 2 &#176;C; hot side: 33 &#177; 2 &#176;C) using an infrared thermometer. 
		NOTE: Just like in the larval test, the heating pad stays on during the whole length of the experiment. Temperature measurements were made at the beginning and end of the experiment. Although the temperature did fluctuate by &#177; 2 &#176;C, there was still at least an 8 &#176;C temperature difference between the hot and cold conditions.</li>
		<li>Place the tube upside down containing one gravid female in the opening in the center of each cross. After the female enters the cross, remove the tube and close the opening with its small lid. After closing all the crosses, place a black cardboard in the front of the shelf to enclose the experimental setup. Let the experiment run for 4 h.</li>
		<li>After that, remove the female, by carefully catching it with a tube, and check if there were any eggs on the substrates.</li>
		<li>Identify the lid of each Petri dish with substrate type from each cross. Use 70% ethanol to clean the crosses (inside the cross and the lid) from any odor from the test. 
		NOTE: In case the eggs cannot be counted right after the experiment, the Petri dishes with substrates can be stored at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Egg count 
	NOTE: If the substrates in the Petri dishes were frozen, thaw them prior to counting.
	<ol>
		<li>Use a stereomicroscope to count the number of eggs laid in each substrate. Use a brush and water to help separate the eggs to count them.</li>
	</ol>
	</li>
</ol>
3. Data analysis and statistics
<ol>
	<li>Preference indexes calculation
	<ol>
		<li>For each replicate of larval (n = 36) and female (n = 30) tests, calculate the Preference Index for meat (designated as PImeat) by determining the ratio of larvae or eggs present on Fresh substrates (Fresh hot and Fresh cold) to the total count of larvae or eggs on all substrates (fresh hot + fresh cold + rotten hot + rotten cold). 
		PImeat = (# larvae or eggs on Fresh substrates) / # Total larvae or eggs 
		&#8203;NOTE: The terms &#34;Hot&#34; and &#34;Cold&#34; are indicative of temperature conditions of 33 &#177; 2 &#176;C and 25 &#177; 2 &#176;C, respectively.</li>
		<li>Likewise, calculate the Preference Index for temperature (PItemp) for each replicate of larval and female tests as the number of larvae or eggs present on Hot substrates (fresh Hot and rotten Hot) divided by the total number of larvae or eggs on all substrates (fresh hot + fresh cold + rotten hot + rotten cold). 
		PItemp = (# larvae or eggs on Hot substrates) / # Total larvae or eggs 
		&#8203;NOTE: Values close to 1 reflect a preference for Fresh or Hot substrates and values close to zero indicate a preference for Rotten or Cold substrates. The PIs can be calculated manually or using the code provided (Supplemental Files S1 and Supplemental Files S2).</li>
	</ol>
	</li>
	<li>Comparison of the observed preference to simulated random data
	<ol>
		<li>Run the provided code (Supplementary File S1 and Supplementary File S1) to generate the simulated data and to compare it with the observed data. 
		NOTE: This code generates 1000 simulated random dataset for larvae and females and calculates the Preference Indexes (PIs) for each replicate of the simulated, and the observed data of L. cuprina. The simulations assume that both larvae and females exhibit no substrate preference and make random choices. The simulations incorporate key behavioral aspects of the animals, encompassing various scenarios such as: the probability of larvae not selecting any substrate, and adult females concentrating their egg-laying on a single substrate or distributing their eggs either uniformly or not among different substrates. Generalized Linear Models (GLM, family: quasibinomial; link: logit) were used to compare the observed data from the behavior assays with the simulated random data. The employed GLM was well-suited for this analysis due to the bounded nature of the Preference Index (PI), ranging between 0 and 1. GLMs are adept at handling non-normally distributed response variables and allow for robust statistical comparisons. This choice facilitated meaningful insights by enabling to effectively compare observed data from behavior assays with complex patterns generated by simulated random data. Minor adjustments to the code may be needed for other structured datasets.</li>
	</ol>
	</li>
</ol>

None declared.

Understanding the evolution of feeding habits, particularly in the context of parasitism in blowflies, requires the examination of substrate preferences throughout different life stages for feeding or oviposition. Therefore, in this study, robust and straightforward methods were proposed for investigating substrate preferences in larvae and females of blowflies. These methods were tested in Lucilia cuprina, a facultative parasitic blowfly2. Interestingly, the experiments unveiled a distin...

Flies, particularly calyptrate muscoids (including blowflies, house flies, bot flies and flesh flies among others), exhibit a wide range of lifestyles, encompassing parasitic and necro-saprophagous behaviors1. Parasitic species typically cause myiasis, an infestation of live tissues by maggots (larvae)2. In the Calliphoridae family, both obligate and facultative parasitic species are major livestock pests responsible for economic losses and poor animal welfare due to maggot infestations2,3,4,5,6,7. Obligate parasites, such as the New World and Old World screwworms (Cochliomyia hominivorax and Chrysomyia bezziana, respectively), are especially problematic4,7,8,9,10 along with facultative parasites, such as the sheep blowflies (Lucilia cuprina and Lucilia sericata)2,5,6,7. Non-parasitic species, including sapro-necrophagous ones, develop in decaying and necrotic organic matter and are commonly found in unsanitary environments. Their strictly non-parasitic lifestyle can be successfully used for maggot therapy, which uses fly larvae to clean wounds of necrotic tissues11,12,13. Blowflies are also used in forensic science, as they are among the first organisms to locate and colonize recently deceased bodies, with the developing larvae serving as a means of estimating the time of death14.
Blowfly lifestyles have been the subject of various research studies (e.g.,15,16,17,18,19,20,21) due to their significance in relation to human interests. Understanding the biological mechanisms governing a species&#39; lifestyle can provide valuable insights into improving methods aimed at controlling pest species. Moreover, the diversity and evolution of blowfly lifestyles offer an ideal context to study the origins and mechanisms of complex traits (e.g., parasitism). Parasitism due to maggots feeding on live tissue has evolved independently several times within the Calliphoridae family22,23. However, the evolutionary history of the feeding habits of blowflies is still largely unknown, with studies restricted to mapping the habits along phylogenies (e.g.,16,19,22) without the aid of functional assays. For instance, it is uncertain whether obligate parasites evolved from generalists (i.e., facultative parasites) or directly from necrophagous species. The molecular, physiological, and behavioral processes accompanying the evolutionary shifts in lifestyle are also largely unknown.
In this context, facultative parasites, such as the sheep blowfly Lucilia cuprina, that can develop as parasites on a host or as necrophages on cadavers offer the possibility to explore the factors and mechanisms controlling lifestyle choices. Lucilia cuprina is a cosmopolitan species known for causing sheep flystrike, especially in Australia where it is considered a pest3,16. Myiasis due to L. cuprina can also occur in other livestock animals, pets, and humans3,24,25,26,27,28,29,30. However, its larvae can also develop in necrotic tissues and decaying matter and this species has been successfully used in forensic entomology as it is very fast to locate and colonize corpses31,32,33,34. Although the parasitic versus non-parasitic lifestyle of blowflies is defined by the larval stage, it is the adult female that selects the oviposition site. Consequently, the adult female heavily influences the larvae&#39;s lifestyle, as the latter have limited mobility. However, the female&#39;s choice does not necessarily imply that the larvae would prefer the same substrate when presented with a choice35. One hypothesis is that behavioral changes leading to females laying their eggs on live tissue could have been part of an early switch towards a parasitic lifestyle. Pre-adaptations or physiological capabilities of the resulting larvae would have been essential for their successful development on the live tissue, leading to the emergence of the parasitic lifestyle. As such, the processes affected and selected may not necessarily align between both life stages.
In this context, two methods were developed to test behavioral preference in blowflies, in particular, for, L. cuprina, regarding larval feeding substrate (larval preference assay) and oviposition site (female preference assay). These methods take into account two interacting factors: temperature and meat freshness. Temperature was chosen as a crucial factor since most cases of myiasis occur in homeothermic animals2. Hence, a temperature of 33 &#176;C was selected as a proxy for the &#34;parasitic lifestyle factor&#34;, whereas a temperature of 25 &#176;C (room temperature) represents the &#34;non-parasitic factor&#34;. A temperature of 25 &#176;C was chosen as it is representative of the average yearly temperature recorded in Brazil (National Institute of Meteorology, INMET). Additionally, two types of meat substrates were considered, both from bovine sources: (i)&#160;fresh meat supplemented with blood, mimicking the substrate for the parasitic lifestyle, which is used to rear the parasitic blowfly Co. hominivorax in laboratory conditions36, and (ii) 5-day-old rotten meat, emulating the substrate for the necrophagous lifestyle. The bovine substrate is commonly used to rear L. cuprina in laboratory conditions27,37,38,39 as it offers several advantages in terms of availability, cost-effectiveness, and practicality while being an ecologically justifiable substrate. Other studies40,41 comparing the effect of rotten versus fresh substrates in blowflies have used 7-day-old rotten substrate (in anaerobic conditions) and showed an adverse effect of the rotten substrate on developmental rates, survival, and growth. As L. cuprina is known to colonize fresh cadavers which are usually exposed to air, we resolved to use 5-day-old rotten meat (ground beef) in non-hermetic pots (aerobic and anaerobic decomposition) to mimic a necrophagous substrate.
The experimental designs presented here offer the advantage of discerning preferences for individual factors as well as their combined effects. Moreover, the phenotypes scored, namely the choice of the larval feeding substrate and the number of eggs laid, are directly relevant to the biological and ecological aspects of blowfly species. The suitability of these protocols is highlighted by demonstrating their effectiveness in L. cuprina. Additionally, a script for statistical analysis is provided, which can be used to compare the observed results obtained in L. cuprina to simulated random data, ensuring robust statistical analysis and interpretations.

<table><tbody><tr><td>Agar</td><td>Sigma-Aldrich</td><td>05038-500G</td><td>For microbiology</td></tr><tr><td>Black cardboards</td><td>-</td><td>-</td><td>70x50 cm</td></tr><tr><td>Bovine blood with anticoagulat&amp;nbsp;</td><td>-</td><td>-</td><td>50% pure bovine blood with anticoagulant (3.8% sodium citrate) + 50% of filtered water</td></tr><tr><td>Bovine ground Meat</td><td>-</td><td>-</td><td>Around 7-8% of fat</td></tr><tr><td>Brush</td><td>-</td><td>-</td><td>Made with plastic</td></tr><tr><td>Conical tube</td><td>Falcon or Generic</td><td>-</td><td>50 mL</td></tr><tr><td>Cross-shaped glass containers</td><td>Handmade</td><td>NA</td><td>48x48 cm, 8 cm of height and 8 cm of width</td></tr><tr><td>Erlenmeyer</td><td>Vidrolabor</td><td>NA</td><td>500 mL</td></tr><tr><td>70% Ethanol</td><td>Synth</td><td>A1084.01.BL</td><td>70% ethyl ethanol absolute + 30% filtered water</td></tr><tr><td>Graduated cylinder</td><td>Nalgon or Generic</td><td>-</td><td>500 mL and 50 mL</td></tr><tr><td>Heating pad</td><td>Thermolux</td><td>-</td><td>30x40 cm dimensions, 40 W, 127 V</td></tr><tr><td>Infrared thermometer</td><td>HeTaiDa</td><td>HTD8808</td><td>Non-contact body thermometer (Sample Rate: 0.5 S,&lt;br/&gt; Accuracy: &amp;plusmn;0.2 &amp;deg;C,&lt;br/&gt; Measuring: 5-15 cm)</td></tr><tr><td>Petri dish (Glass)</td><td>Precision</td><td>NA</td><td>150x20 mm dimensions</td></tr><tr><td>&amp;nbsp; &amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;&amp;nbsp;</td><td>(Note: the petri dishes can be plastic if used only once)</td></tr><tr><td>Petri dish PS</td><td>Cralplast</td><td>18130</td><td>60x15 mm dimensions</td></tr><tr><td>Plastic Pasteur pipette</td><td>-</td><td>-</td><td>3 mL (total volume)</td></tr><tr><td>Sodium citrate</td><td>Synth</td><td>C11033.01.AG</td><td>3.8% Sodium citrate (38 g diluted in 1L of filtered water)</td></tr><tr><td>Spoons</td><td>-</td><td>-</td><td>More than one spoon is necessary. Use one for each type of meat substrate. Preferably stainless steel.</td></tr><tr><td>Stainless steel spatula</td><td>Generic</td><td>-</td><td>Flat end and spoon end</td></tr><tr><td>Stereomicroscope</td><td>Bioptika</td><td>-</td><td>WF10X/22 lenses</td></tr><tr><td>Tweezer</td><td>-</td><td>-</td><td>Metal made and fine point</td></tr><tr><td>White led light strips</td><td>NA</td><td>NA</td><td>4.8 W, 2x0.05 mm&amp;sup2;, 320 lumens, Color temperature:6500 K (white)</td></tr></tbody></table>

exploring life history choices: using temperature and substrate type as interacting factors for blowfly larval and female preferences

Blowflies (Diptera: Calliphoridae) present a wide range of larval lifestyles, typically classified as obligate parasitism, facultative parasitism, and complete sapro-necrophagy. Several parasitic species, both obligate and facultative, are considered to be of sanitary and economic importance, as their larvae can cause myiasis (maggot infestation in live tissue). However, it is noteworthy that the adult female plays a decisive role as she chooses the oviposition site, and, therefore, largely determines the feeding habit and developmental conditions of the larvae. In this study, two protocols are proposed to test larval feeding preference and female oviposition site preference considering two interacting factors: meat substrate type and temperature. The setups presented here allowed to test Lucilia cuprina larvae and gravid females in a four-choice assay with two temperatures (33 &plusmn; 2 &#176;C and 25 &plusmn; 2 &#176;C) and two types of meat substrates (fresh meat supplemented with blood and 5-day-old rotten meat). Larvae or gravid females can choose to burrow or lay their eggs, respectively, in either of the following: rotten meat at 25 &#176;C (simulating a necrophagous species condition), fresh meat supplemented with blood at 33 &#176;C (simulating a parasitic species condition), and two controls, rotten meat at 33 &#176;C, or fresh meat supplemented with blood at 25 &#176;C. The preference is assessed by counting the number of larvae or eggs laid in each option for each replicate. Comparing the observed results to a random distribution allowed for the estimation of the statistical significance of the preference. The results indicated that L. cuprina larvae have a strong preference for the rotten substrate at 25 &#176;C. Conversely, oviposition-site preference by females was more varied for the meat type. This methodology can be adapted to test the preference of other insect species of similar size. Other questions can also be explored by using alternative conditions.

To demonstrate the effectiveness of the presented methods, the experiments were conducted using a laboratory population of Lucilia cuprina (family: Calliphoridae), a facultative parasitic blowfly2. The entire raw dataset obtained for this species can be found in Supplementary File S3 with the results for the larval and female substrate preference tests. To assess if the larvae and females display a preference for any substrate, the observed data were compared to 1000 simu...

Watch this Scientific Journal Video about Exploring Life History Choices: Using Temperature and Substrate Type as Interacting Factors for Blowfly Larval and Female Preferences at JoVE.com

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

Herein, two protocols for assessing food source and oviposition preferences in larvae and females of blowflies are detailed. These comprise four choices with two interacting factors: substrate type and temperature. The assays enable the determination of the food source preference of the larvae and the oviposition site preference for the females.

Blowflies (Diptera: Calliphoridae) present a wide range of larval lifestyles, typically classified as obligate parasitism, facultative parasitism, and ...

exploring-life-history-choices-using-temperature-substrate-type-as

Research

JoVE Journal

Behavior

1.3K Views.  University of São Paulo. Bromvliegen zijn een diverse groep vliegen die obligate of facultatieve parasieten kunnen zijn, evenals sapro-necrofaag. Obligate parasitaire soorten voeden zich uitsluitend met levende weefsels en veroorzaken myiasis bij gewervelde dieren, terwijl sapro-necrofaag soorten zich voeden met rottend organisch materiaal. Facultatieve parasieten kunnen zich voeden met zowel levend als rottend materiaal en al dan niet als parasiet leven.Hoewel het larvale stadium het meest problematisch is, s...

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

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Neuroscience

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Here we present a protocol to automatically determine the locomotor performance of Drosophila at changing temperatures using a programmable temperature-controlled arena that produces fast and accurate temperature changes in time and space.

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

A Wind Tunnel for Odor Mediated Insect Behavioural Assays

Olfaction is the most important sensory mechanism by which many insects interact with their environment and a wind tunnel is an excellent tool to study insect chemical ecology. Insects can locate point sources in a three-dimensional environment through the sensory interaction and sophisticated behavior. The quantification of this behavior is a key element in the development of new tools for pest control and decision support. A wind tunnel with a suitable flight section with laminar air flow, visual cues for in-flight feedback and a variety of options for the application of odors can be used to measure complex behaviour which subsequently may allow the identification of attractive or repellent odors, insect flight characteristics, visual-odor interactions and interactions between attractants and odors lingering as background odors in the environment. A wind tunnel holds the advantage of studying the odor mediated behavioural repertoire of an insect in a laboratory setting. Behavioural measures in a controlled setting provide the link between the insect physiology and field application. A wind tunnel must be a flexible tool and should easily support the changes to setup and hardware to fit different research questions. The major disadvantage to the wind tunnel setup described here, is the clean odor background which necessitates special attention when developing a synthetic volatile blend for field application.

Here, we describe the construction and use of a wind tunnel for odor mediated behavioural assays with insects. The wind tunnel design facilitates the release of odor sources by several methods, with and without visual stimuli. Wind tunnel experiments are important methods to identify behaviorally active volatile chemicals.

Olfaction is the most important sensory mechanism by which many insects interact with their environment and a wind tunnel is an excellent tool to study ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Adapting Microrespirometry for Drosophila - Insights into CASK Mutant Metabolism

Measuring O2 Consumption in Drosophila melanogaster Using Coulometric Microrespirometry

Coulometric microrespirometry is a straightforward, inexpensive method for measuring the O2 consumption of small organisms while maintaining a stable environment. A coulometric microrespirometer consists of an airtight chamber in which O2 is consumed and the CO2 produced by the organism is removed by an absorbent medium. The resulting pressure decrease triggers electrolytic O2 production, and the amount of O2 produced is measured by recording the amount of charge used to generate it. In the present study, the method has been adapted to Drosophila melanogaster tested in small groups, with the sensitivity of the apparatus and the environmental conditions optimized for high stability. The amount of O2 consumed by wildtype flies in this apparatus is consistent with that measured by previous studies. Mass-specific O2 consumption by CASK mutants, which are smaller and known to be less active, was not different from congenic controls. However, the small size of CASK mutants resulted in a significant reduction in O2 consumption on a per-fly basis. Therefore, the microrespirometer is capable of measuring O2 consumption in D. melanogaster, can distinguish modest differences between genotypes, and adds a versatile tool for measuring metabolic rates.

Author Spotlight: Influence of Temperature on Drosophila melanogaster and Desert-Adapted Beetles 

Coulometric respirometry is ideal for measuring the metabolic rate of small organisms. When adapted for Drosophila melanogaster in the present study, measured O2 consumption was within the range reported for wildtype D. melanogaster by previous studies. Per-fly O2 consumption by CASK mutants, which are smaller and less active, was significantly lower than the wildtype.

Coulometric microrespirometry is a straightforward, inexpensive method for measuring the O2 consumption of small organisms while maintaining a stable ...

Determining Temperature Preference of Mosquitoes and Other Ectotherms

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on their fitness, as well as other characteristics. Consequently, many such ectotherms seek out their optimal temperature range. Although temperature preferences of mosquitoes and other insects have been well studied, the traditional experimental setup is performed using a temperature gradient on an aluminum surface in a highly enclosed space. In some cases, this equipment restricts many natural behaviors, such as flying, which may be important in preference selection.
The objective of this study is to observe insect preference for air temperature by using a two-chamber apparatus with sufficient room for flight. The two chambers consist of independent temperature-controlled incubators, each with a large aperture. The incubators are connected by these apertures using a short acrylic bridge. Inside the incubators are two netted cages, linked via the apertures and bridge, allowing the insects to freely fly between the different conditions. The acrylic bridge also acts as a temperature gradient between the two incubators.
Due to the spacious area in the cage and easy construction, this method can be used to study any small ectotherm and/or any manipulation which may alter temperature preference including sensory organ manipulation, diet, gut flora, and endosymbiont presence at biosafety levels 1 or 2 (BSL 1 or 2). Additionally, the apparatus can be used for the study of pathogen infection using further containment (e.g., inside of a biosafety cabinet) at BSL 3.

Insects have an optimal environmental temperature range which they seek to remain within, and many external and internal factors can alter this preference. Here, we describe a cost-effective and simple method to study temperature choice, which allows insects to freely exhibit their natural behaviors.

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on ...

Biology

Bradysia coprophila Lab Protocols and Chromosomal Research

Laboratory Maintenance of the Lower Dipteran Fly Bradysia (Sciara) coprophila: A New/Old Emerging Model Organism

Laboratory stocks of the lower dipteran fly, Bradysia (Sciara) coprophila, have been maintained for over a century. Protocols for laboratory upkeep of B. coprophila are presented here. These protocols will be useful for the rapidly increasing number of laboratories studying B. coprophila to take advantage of its unique biological features, which include (1) a monopolar spindle in male meiosis I; (2) non-disjunction of the X dyad in male meiosis II; (3) chromosome imprinting to distinguish maternal from paternal homologs; (4) germ line-limited (L) chromosomes; (5) chromosome elimination (paternal chromosomes in male meiosis I; one to two X chromosomes in early embryos; L chromosomes from the soma in early embryos); (6) sex determination by the mother (there is no Y chromosome); and (7) developmentally regulated DNA amplification at the DNA puff loci in larval salivary gland polytene chromosomes. 
It is now possible to explore these many unique features of chromosome mechanics by using the recent advances in sequencing and assembly of the B. coprophila genome and the development of transformation methodology for genomic engineering. The growing scientific community that uses B. coprophila for research will benefit from the protocols described here for mating the flies (phenotypic markers for mothers that will have only sons or only daughters; details of mass mating for biochemical experiments), checking embryo hatch, feeding larvae, and other comments on its rearing.

Author Spotlight: Exploring Bradysia coprophila's Unique Biology &#8211; A Guide to Laboratory Maintenance

This paper outlines the laboratory maintenance (including mating and feeding) of the lower dipteran fly Bradysia (Sciara) coprophila.

Laboratory stocks of the lower dipteran fly, Bradysia (Sciara) coprophila, have been maintained for over a century. Protocols for laboratory upkeep of B. ...

Temperature Gradient Assay: A Method to Test Temperature Preference in Drosophila Larvae

Source: Liu, J., et al. <a href="https://www.jove.com/video/57963/" target="_blank">A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae</a>. J. Vis. Exp. (2018).
Drosophila fruit flies can distinguish small changes in temperature and therefore select environments with favorable thermal conditions. This video describes a behavioral assay that tests the temperature preference of Drosophila larvae on a linear thermal gradient.

Source: Liu, J., et al. A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae. J. Vis. Exp. (2018).
Drosophila fruit flies ...

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

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