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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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, 
			Accuracy: &#177;0.2 &#176;C, 
			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>&#160; &#160;</td>
			<td>&#160; &#160;</td>
			<td>&#160; &#160;&#160;</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&#178;, 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

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Behavior

1.2K Views.  University of São Paulo. 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. 

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

The Forced Swim Test as a Model of Depressive-like Behavior

The goal of the present protocol is to describe the forced swim test (FST), which is one of the most commonly used assays for the study of depressive-like behavior in rodents. The FST is based on the assumption that when placing an animal in a container filled with water, it will first make efforts to escape but eventually will exhibit immobility that may be considered to reflect a measure of behavioral despair. This test has been extensively used because it involves the exposure of the animals to stress, which was shown to have a role in the tendency for major depression. Additionally, the FST has been shown to share some of the factors that are influenced or altered by depression in humans, including changes in food consumption, sleep abnormalities and drug-withdrawal-induced anhedonia. The main advantages of this procedure are that it is relatively easy to perform and that its results are easily and quickly analyzed. Moreover, its sensitivity to a broad range of antidepressant drugs that makes it a suitable screening test is one of the most important features leading to its high predictive validity. Despite its appeal, this model has a number of disadvantages. First, the issue of chronic augmentation is problematic in this test because in real life patients need to be treated for at least several weeks before they experience any relief from their symptoms. Last, due to the aversiveness of the FST, it is important to take into account possible influences it might have on brain structure/function if brain analyses are to be carried out following this procedure.

This protocol describes the forced swim test, which is used for the study of depressive-like behavior in rodents. This procedure involves placing an animal in a container filled with water that eventually will lead to the exhibition of immobility behavior, which is considered to reflect behavioral despair.

The goal of the present protocol is to describe the forced swim test (FST), which is one of the most commonly used assays for the study of depressive-like ...

Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the necessary equipment is now available by several commercial companies. This protocol provides a detailed description of a two-way shuttle-box avoidance learning paradigm in rodents (here Mongolian gerbils; Meriones unguiculatus) in combination with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical electrophysiological in vivo recordings. The detailed protocol is applicable to study multiple aspects of learning behavior and perception in different rodent species.
Site-specific ICMS of auditory cortical circuits as conditioned stimuli here is used as a tool to test the perceptual relevance of specific afferent, efferent and intracortical connections. Distinct activation patterns can be evoked by using different stimulation electrode arrays for local, layer-dependent ICMS or distant ICMS sites. Utilizing behavioral signal detection analysis it can be determined which stimulation strategy is most effective for eliciting a behaviorally detectable and salient signal. Further, parallel multichannel-recordings using different electrode designs (surface electrodes, depth electrodes, etc.) allow for investigating neuronal observables over the time course of such learning processes. It will be discussed how changes of the behavioral design can increase the cognitive complexity (e.g. detection, discrimination, reversal learning).

Shuttle-box avoidance learning is well-established in behavioral neuroscience. This protocol describes how shuttle-box learning in rodents can be combined with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical in vivo recordings as a tool to study multiple aspects of learning and perception.

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the ...

Determining Ultrasonic Vocalization Preferences in Mice using a Two-choice Playback Test

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, sex, condition, and genetic background of the emitting animal. Although many studies have characterized these differences, whether receiver mice can discriminate among objectively different USVs and show preferences for particular sound traits remains to be elucidated. To determine whether mice can discriminate between different characteristics of USVs, a playback experiment was developed recently, in which preference responses of mice to two different USVs could be evaluated in the form of a place preference.
First, USVs from mice were recorded. Then, the recorded USVs were edited, trimmed accordingly, and exported as stereophonic sound files. Next, the USV amplitudes generated by the two ultrasound emitters used in the experiment were adjusted to the same sound pressure level. Nanocrystalline silicon thermo-acoustic emitters were used to play the USVs back. Finally, to investigate the preference of subject mice to selected USVs, pairs of two differing USV signals were played back simultaneously in a two-choice test box. By repeatedly entering a defined zone near an ultrasound emitter and searching the wire mesh in front of the emitter, the mouse reveals its preference for one sound over another. This model allows comparing the attractiveness of the various features of mouse USVs, in various contexts.

Ultrasonic vocalizations (USVs) in mice differ depending on age, sex, condition, and genetic background. Using two ultrasound emitters broadcasting simultaneously in different locations, this two-choice test can evaluate murine recognition and preference responses to different characteristics of USVs.

Mice emit ultrasonic vocalizations (USVs) during a variety of conditions, such as pup isolation and adult social interactions. These USVs differ with age, ...

Psychophysical Tracking Method to Measure Taste Preferences in Children and Adults

The Monell two-series, forced-choice, paired-comparison tracking method provides a reliable measure of sweet taste preferences from childhood to adulthood. The method, which is identical for children, adolescents, and adults, is of short duration (&#60; 15 min), does not rely on sustained attention or place demands on memory (which would yield spurious age differences), and minimizes the impact of language development, making this method amenable to the cognitive limitations of pediatric populations. In this whole-mouth tasting method, subjects are asked to taste (without swallowing) pairs of solutions of different sucrose concentrations and to point to the solution they prefer. Each subsequent pair contains the participant&#39;s preceding preferred concentration and an adjacent stimulus concentration. The procedure continues until the subject chooses either a given concentration of sucrose when paired with both a higher and a lower concentration, or the highest or lowest concentration two consecutive times. Subjects are prevented from reaching response criteria on the basis of first or second position bias by the two-series design of the method, which counterbalances the order of solution presentation within each pair between the series (the weaker concentration is presented first in Series 1, second in Series 2). The geometric mean of the two sucrose concentrations chosen in Series 1 and 2 is an estimate of the participant&#39;s most preferred level of sucrose. Sucrose preference as determined with this laboratory-based measure has been shown to be associated with preference for sugars in foods and beverages and with taste receptor genotype, family history of alcoholism, and race/ethnicity, as well as depressive symptomatology among pediatric populations. The method has real-world relevance and has been applied to determine most preferred level of other tastes (e.g., salt), making it a valuable psychophysical tool.

Sweet taste has powerful hedonic appeal among people of all ages, particularly children. Described herein is a reliable and valid method that can be used to determine the level of sweetness most preferred, making it a valuable psychophysical tool for scientists.

The Monell two-series, forced-choice, paired-comparison tracking method provides a reliable measure of sweet taste preferences from childhood to ...

Methodology for Establishing a Community-Wide Life Laboratory for Capturing Unobtrusive and Continuous Remote Activity and Health Data

An end-to-end suite of technologies has been established for the unobtrusive and continuous monitoring of health and activity changes occurring in the daily life of older adults over extended periods of time. The technology is aggregated into a system that incorporates the principles of being minimally obtrusive, while generating secure, privacy protected, continuous objective data in real-world (home-based) settings for months to years. The system includes passive infrared presence sensors placed throughout the home, door contact sensors installed on exterior doors, connected physiological monitoring devices (such as scales), medication boxes, and wearable actigraphs. Driving sensors are also installed in participants' cars and computer (PC, tablet or smartphone) use is tracked. Data is annotated via frequent online self-report options that provide vital information with regard to the data that is difficult to infer via sensors such as internal states (e.g., pain, mood, loneliness), as well as data referent to activity pattern interpretation (e.g., visitors, rearranged furniture). Algorithms have been developed using the data obtained to identify functional domains key to health or disease activity monitoring, including mobility (e.g., room transitions, steps, gait speed), physiologic function (e.g., weight, body mass index, pulse), sleep behaviors (e.g., sleep time, trips to the bathroom at night), medication adherence (e.g., missed doses), social engagement (e.g., time spent out of home, time couples spend together), and cognitive function (e.g., time on computer, mouse movements, characteristics of online form completion, driving ability). Change detection of these functions provides a sensitive marker for the application in health surveillance of acute illnesses (e.g., viral epidemic) to the early detection of prodromal dementia syndromes. The system is particularly suitable for monitoring the efficacy of clinical interventions in natural history studies of geriatric syndromes and in clinical trials.

Unobtrusive sensors and pervasive computing technology incorporated into the daily home life of older adults enables meaningful health and activity changes to be recorded continuously for months to years, providing ecologically valid, high frequency, multi-domain data for research or clinical use.

An end-to-end suite of technologies has been established for the unobtrusive and continuous monitoring of health and activity changes occurring in the ...

Assessment of Social Transmission of Food Preferences Behaviors

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy aging groups. For example, olfactory dysfunction in AD can present as impairment in olfactory recognition, emerging during early stages of the disease and worsening while the disease progresses. The social transmission of food preferences (STFP) task is based on a rudimentary form of communication between rodents concerning distant foods dependent on the transmission of olfactory cues. Healthy wild-type mice would prefer to eat a novel, flavored food that was previously cued by a conspecific, and this food preference would be hampered in transgenic AD mice, such as the APP/PS1 model. Indeed, a strong preference for the cued food in C57Bl6/J mice of 3 months of age was found, and this was reduced in 3 months old transgenic APP/PS1 mice. In summary, STFP task could be a powerful measure to be integrated in present subclinical detection assays of AD.

This paper presents a protocol for the investigation of social transmission of food preference in mice. The advantages and possible applications for this procedure, for instance, in detecting early changes in AD mouse models, are highlighted. To conclude, interpretation of the results in light of critical details are discussed.

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy ...

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 ...

Exploring Infant Sensitivity to Visual Language using Eye Tracking and the Preferential Looking Paradigm

We discuss the use of the preferential looking paradigm in eye tracking studies in order to study how infants develop, understand, and attend to the world around them. Eye tracking is a safe and non-invasive way to collect gaze data from infants, and the preferential looking paradigm is simple to design and only requires the infant to be attending to the screen. By simultaneously showing two visual stimuli that differ in one dimension, we can assess whether infants show different looking behavior for either stimulus, thus demonstrating sensitivity to that difference. The challenges in such experimental approaches are that experiments must be kept brief (no more than 10 min) and be carefully controlled such that the two stimuli differ in only one way. The interpretation of null results must also be carefully considered. In this paper, we illustrate a successful example of an infant eye tracking study with a preferential looking paradigm to discover that 6-month-olds are sensitive to linguistic cues in a signed language despite having no prior exposure to signed language, suggesting that infants possess intrinsic or innate sensitivities to these cues.

Eye tracking studies using a preferential looking paradigm can be used to study infants&#39;&#160;emerging understanding of, and attention to, their external visual world.

We discuss the use of the preferential looking paradigm in eye tracking studies in order to study how infants develop, understand, and attend to the world ...

A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice

Sleep disturbance is generally common in populations as a chronic disease or a complained event. Chronic sleep disturbance is proposed to be closely linked to the pathogenesis of diseases, especially neurodegenerative diseases. We recently found that 2 months of sleep fragmentation initiated Alzheimer&#8217;s disease (AD)-like behavioral and pathological changes in young wild-type mice. Herein, we present a standardized protocol to achieve chronic sleep fragmentation (CSF). Briefly, CSF was induced by an orbital rotor vibrating at 110 rpm and operating with a repetitive cycle of 10 s-on, 110 s-off, during light-ON phase (8:00 AM&#8211;8:00 PM) continuously for up to 2 months. Impairments of spatial learning and memory, anxiety-like but not depression-like behavior in mice as consequences of CSF modeling, were evaluated with Morris water maze (MWM), Novel object recognition (NOR), Open field test (OFT) and Forced swimming test (FST). In comparison with other sleep manipulations, this protocol minimizes the handling labors and maximizes the modeling efficiency. It produces stable phenotypes in young wild-type mice and can be potentially generated for a variety of research purposes.

Presented here is a protocol for chronic sleep fragmentation (CSF) model achieved by an electrically controlled orbital rotor, which could induce confirmed cognitive deficit and anxiety-like behavior in young wild-type mice. This model can be applied to explore the pathogenesis of chronic sleep disturbance and related disorders.

Sleep disturbance is generally common in populations as a chronic disease or a complained event. Chronic sleep disturbance is proposed to be closely ...

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. Animals cope with these stimuli by modifying their behavior. Understanding the mechanisms underlying such behavioral modification requires quantification of neural activity during the behavior of interest. Here, we report a method for calcium imaging in freely behaving Caenorhabditis elegans with nonlocalized vibration of specific frequency, displacement, and duration. This method allows the production of well-controlled, nonlocalized vibration using an acoustic transducer and quantification of evoked calcium responses at single-cell resolution. As a proof of principle, the calcium response of a single interneuron, AVA, during the escape response of C. elegans to vibration is demonstrated. This system will facilitate understanding of neural mechanisms underlying behavioral responses to mechanical stimuli.

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Reported here is a system for calcium imaging in freely behaving Caenorhabditis elegans with well-controlled, nonlocalized vibration. This system allows researchers to evoke nonlocalized vibrations with well-controlled properties at nano-scale displacement and to quantify calcium currents during responses of C. elegans to the vibrations.

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. ...