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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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 ± 2 °C and 25 ± 2 °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 °C (simulating a necrophagous species condition), fresh meat supplemented with blood at 33 °C (simulating a parasitic species condition), and two controls, rotten meat at 33 °C, or fresh meat supplemented with blood at 25 °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 °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.

Introduction

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' 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's lifestyle, as the latter have limited mobility. However, the female'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 °C was selected as a proxy for the "parasitic lifestyle factor", whereas a temperature of 25 °C (room temperature) represents the "non-parasitic factor". A temperature of 25 °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) 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.

Protocol

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

  1. Preparation of the Petri dishes containing 2% agar
    1. 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.
    2. 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.
  2. Preparation of substrates
    1. 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.
    2. 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 °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.
    3. 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.
  3. Experimental setup
    1. At a room temperature (RT) of 25 °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).
    2. 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.
    3. 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.
  4. Larval test
    1. Check the temperature of the substrates (cold side: 25 ± 2 °C; hot side: 33 ± 2 °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 ± 2 °C, there was still at least an 8 °C temperature difference between the hot and cold conditions.
    2. 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.
    3. 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.
      ​NOTE: Lucilia cuprina larvae stay in their chosen substrate, as observed in this experiment.

2. Female oviposition site preference

  1. Experimental setup
    1. 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.
    2. At RT (25 °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.
    3. Cover the heating pad and leveling paperboard pads with a black cardboard to keep the same visual pattern below all the substrates.
    4. 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.
    5. Use 70% ethanol to clean the crosses (inside the cross and the lid) to avoid odor contamination.
  2. Preparation of substrates
    1. 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.
    2. 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.
    3. 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'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.
  3. Female test
    1. 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.
    2. Check the temperature of the substrates in the crosses (cold side: 25 ± 2 °C; hot side: 33 ± 2 °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 ± 2 °C, there was still at least an 8 °C temperature difference between the hot and cold conditions.
    3. 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.
    4. After that, remove the female, by carefully catching it with a tube, and check if there were any eggs on the substrates.
    5. 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 °C.
  4. Egg count
    NOTE: If the substrates in the Petri dishes were frozen, thaw them prior to counting.
    1. 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.

3. Data analysis and statistics

  1. Preference indexes calculation
    1. 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
      ​NOTE: The terms "Hot" and "Cold" are indicative of temperature conditions of 33 ± 2 °C and 25 ± 2 °C, respectively.
    2. 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
      ​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).
  2. Comparison of the observed preference to simulated random data
    1. 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.

Results

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...

Discussion

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...

Disclosures

None declared.

Acknowledgements

We acknowledge Patrícia 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-São 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).

Materials

NameCompanyCatalog NumberComments
AgarSigma-Aldrich05038-500GFor microbiology
Black cardboards--70x50 cm
Bovine blood with anticoagulat --50% pure bovine blood with anticoagulant (3.8% sodium citrate) + 50% of filtered water
Bovine ground Meat--Around 7-8% of fat
Brush--Made with plastic
Conical tubeFalcon or Generic-50 mL
Cross-shaped glass containersHandmadeNA48x48 cm, 8 cm of height and 8 cm of width
ErlenmeyerVidrolaborNA500 mL
70% EthanolSynthA1084.01.BL70% ethyl ethanol absolute + 30% filtered water
Graduated cylinderNalgon or Generic-500 mL and 50 mL
Heating padThermolux-30x40 cm dimensions, 40 W, 127 V
Infrared thermometerHeTaiDaHTD8808Non-contact body thermometer (Sample Rate: 0.5 S,
Accuracy: ±0.2 °C,
Measuring: 5-15 cm)
Petri dish (Glass)PrecisionNA150x20 mm dimensions
          (Note: the petri dishes can be plastic if used only once)
Petri dish PSCralplast1813060x15 mm dimensions
Plastic Pasteur pipette--3 mL (total volume)
Sodium citrateSynthC11033.01.AG3.8% Sodium citrate (38 g diluted in 1L of filtered water)
Spoons--More than one spoon is necessary. Use one for each type of meat substrate. Preferably stainless steel.
Stainless steel spatulaGeneric-Flat end and spoon end
StereomicroscopeBioptika-WF10X/22 lenses
Tweezer--Metal made and fine point
White led light stripsNANA4.8 W, 2x0.05 mm², 320 lumens, Color temperature:6500 K (white)

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