Name: exploring life history choices: using temperature and substrate type as interacting factors for blowfly larval and female preferences
Uploaded: 2023-11-17T13:30:00
Description: 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

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 Petr...

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 ...

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