Development of the house fly, Musca domestica L. (Diptera: Muscidae), on pork tissue at two temperatures
Presented at the 75th Annual Scientific Conference of the American Academy of Forensic Sciences, February 13–18, 2023, in Orlando, FL.
Abstract
The house fly, Musca domestica, L. (Diptera: Muscidae), is a filth fly that is often associated with criminal and civil investigations surrounding abuse, neglect, and death of humans and other vertebrates. However, development data, which are crucial for determining the age of immatures collected under forensically relevant circumstances, are limited. Given the lack of data and the recognition of population-specific growth patterns, the aim of this study was to generate data for development of a M. domestica population from Texas, USA, on decomposing lean pork at 24.0°C (i.e., approximate room temperature in Texas) and 37.0°C (i.e., approximate human body temperature). As expected, fly development significantly differed between temperatures with development at the higher temperature taking significantly less time (development from egg to adult emergence occurred c. 48.5% faster at 37.0°C than at 24.0°C). The value of this dataset is demonstrated through an applied comparison with previously published data for the house fly. Differences in development times across life stages for the studies are evident, with shorter time of colonization estimations using the data published by Wang et al. (2018), especially in later life stages. These data represent the first development dataset for the house fly on decomposing flesh in North America. Furthermore, the comparison with the previously published dataset demonstrate data from this study are of value for future forensic investigations in Texas or possibly other parts of the United States where this species is encountered, as they can be used to determine time of colonization.
Highlights
- The first known published development study in North America focusing on Musca domestica.
- Highlights possible impact of spatial variation on development rate and therefore TOC estimations.
- Data generated can be applied to forensic investigations where house fly immatures are present.
- Data are relevant to abuse/neglect/death scenarios with average room or human body temperatures.
1 INTRODUCTION
Arthropods are recognized as valuable evidence associated with forensic investigations [1]. In most cases, blow flies (Diptera: Calliphoridae) [2, 3], flesh flies (Diptera: Sarcophagidae) [4], and filth flies (Diptera: Muscidae) [5, 6] are utilized to determine time of colonization [7] as related to a minimum postmortem interval, provided certain assumptions are met [8]. Blow fly species are the most well-studied when it comes to forensically relevant literature [2, 9-12] due to their primary colonization behaviors and their often being abundant with decomposing remains [13]. However, many other species also have the potential to provide valuable data associated with forensic investigations as related to time of colonization (TOC) or other forensically relevant information such as abiotic condition surrounding death [14]. Especially in forensic cases with specific circumstances, such as living victims of abuse or neglect as well as decedents who may have been colonized prior to death, non-calliphorid species can be present and potentially the primary colonizing taxa. Unfortunately, development data are not available for many of these species, thus limiting their evidentiary values.
The house fly, Musca domestica, L. (Diptera: Muscidae), is a cosmopolitan, synanthropic species [15]. While a majority of research on this species is with regard to its status as a health and agriculture nuisance [16, 17] due to its ability to serve as a vector for numerous pathogens [18-20], and its association with confined animal facilities [21, 22], this species also is known to colonize dead [23] or living [24] humans and other vertebrates. In a forensic context, M. domestica has been studied and found in association with decomposing vertebrate remains in China [25], Malaysia [23, 26], Italy [27], and the United States (CAF, JRC, JKT, unpublished data).
It is generally understood that house flies and other Muscidae arrive at decomposing vertebrate remains secondarily to blow flies or flesh flies [13] but under certain circumstances, such as myiasis (e.g., colonization of living tissue) associated with fecal matter, house flies may be the primary and/or sole colonizer, highlighting the importance and need of development data for this species. In such instances, their presence can be used to determine minimal duration of abuse or neglect; however, due to a lack of development data in North America, determining a reliable time of colonization is challenging and leads to numerous assumptions being made [8]. The aims of this study were to (1) determine the development rates of immature M. domestica on pork at two different temperatures selected for their forensic relevance (24°C to represent average room temperature in Texas, USA, for this synanthropic species, and 37°C to represent the average adult human body temperature for cases of myiasis) and (2) compare resulting data with previously published study on house fly development. Data generated will be beneficial in future investigations where immature house flies or associated artifacts are associated with living or deceased humans or other vertebrates, as such information can inform the duration of colonization as related to abuse, neglect, or even a minimum postmortem interval.
2 METHODS
2.1 House fly colony and tissue source
House fly larvae used for this experiment were obtained from a colony maintained at the Forensic Laboratory for Investigative Entomological Sciences (F.L.I.E.S.) Facility at Texas A&M University, College Station, TX, USA, which originated from a population maintained Stephenville, TX, USA. Adult house flies were housed in 30 × 30 × 30 cm BioQuip Bug dorms (BioQuip Inc., CA, USA) located in a rearing room with environmental conditions maintained at 23.8°C, and a 14:10 h L:D cycle. Adults were provided with a 1:1 sugar: powdered milk mixture and water immediately after emergence, which was replenished ad libitum. Adults at c. 9-days-old were provided with 50 g Gainesville diet (50% wheat bran, 30% alfalfa meal, and 20% corn meal) [28] saturated with reverse osmosis water, wrapped in a KimWipe (Kimberly-Clark Corp., Irving, TX, USA) as an oviposition site. Lean boneless pork chops were obtained from H-E-B (Kerrville, TX, USA), partitioned into 50 g samples, placed in a 16.5 × 14.9 cm Ziploc® sandwich bag (S.C. Johnson & Son, Racine, WI, USA), and stored in a −20°C freezer until used.
2.2 Laboratory experiment design
The development study was conducted in two model 136LLVL Percival incubators (Percival Scientific, Inc., Perry, IA, USA), set to 24°C and 37°C, 70% relative humidity (RH), and a 14:10 light–dark cycle. An Onset HOBO MX1104 (Onset, Pocasset, MA, USA) was placed in each growth chamber, and temperature, RH, and light intensity were recorded every 30 s throughout the duration of the study. The percival units were rotated between trials. Data from the thermal monitoring units indicated actual temperatures in the incubators were 23.76 ± 0.28°C and 36.57 ± 0.21°C throughout the study.
The pre-portioned pork tissue in a Ziploc® bag was removed from the freezer 12 h before the study and placed in the growth chamber to allow the tissue to warm to the designated experiment temperature. A 50 g piece of pork was placed on the surface of 225 g of sand (Quikrete Premium Play Sand, Atlanta, GA, USA) in individual 1.10-L styrene mosquito-breeding containers (BioQuip Products, Rancho Dominguez, CA, USA). This was repeated five more times for a total of six containers. Three containers were designated for each temperature treatment. Fly colonies were monitored hourly for oviposition. Eggs <1-h-old were weighed in groups of 10 to obtain an average egg weight, and ~200 egg aliquots were placed on the pork tissue in each replicate. Aliquots of 200 eggs were used based on average eggs produced per oviposition event [29], sampling methods used in this study, and to avoid overcrowding, as overcrowding can lead to larval mortality. After eggs were placed on the diet, the containers were not covered to allow for sufficient air flow and were placed in a randomized location within each incubator determined by a random number generator.
2.3 Life-history traits
Eggs were checked hourly for eclosion. Once initial eclosion was observed for each of the three containers within a temperature treatment, larval collections were made every 4 h. During each collection, three larvae were subsampled unsystematically from each of the six containers, along with recording the time, date, and growth chamber temperature. During a 24-h period, a total of 18 larvae were collected from each individual container (three larvae collected during six time points). Larvae were parboiled for 10 s and then placed in 70% ethanol until analysis. Once larvae began wandering away from the food source (a behavioral change that can be used to separate the feeding third instar and the post-feeding third-instar stages), collections ceased and visual observations were made every 6 h through the duration of the pupal stage until adult emergence. Three trials of this study were conducted at each temperature. For each time point of collection (e.g., 12 h post eclosion at 37°C), there were 27 larvae collected to represent that specific age across the study (three larvae collected from each rearing container, three rearing containers per trial, and three trials conducted).
2.4 Sample processing
All samples were evaluated using a Zeiss Stemi DV4 stereomicroscope (Dublin, CA, USA) at 32× magnification to identify which development stage the individuals were in at that time. Minimum times to reach stage (Table 1) were recorded as the first time point in which the life stage was observed in a sample. Maximum times to complete stage (Table 2) were recorded as the time point following the last sample in which the life stage was observed. Maximum development data were not recorded for the egg and pupal stages.
| Stage | 24°C | 37°C | Statistical significance |
|---|---|---|---|
| First instar | 18.67 ± 0.54 | 9.06 ± 0.19 | F = 284.36; df = 1, 17; p < 0.0001 |
| Second instar | 53.78 ± 2.19 | 25.61 ± 0.79 | F = 146.49; df = 1, 17; p < 0.0001 |
| Third instar | 91.56 ± 2.59 | 40.5 ± 1.01 | F = 338.13; df = 1, 17; p < 0.0001 |
| Post-feeding third instar | 158.57 ± 5.52 | 72.94 ± 1.54 | F = 278.59; df = 1, 15; p < 0.0001 |
| Pupa | 176.89 ± 4.84 | 84.06 ± 3.73 | F = 230.84; df = 1, 17; p < 0.0001 |
| Adult | 334.44 ± 5.19 | 172.06 ± 2.54 | F = 790.03; df = 1,17; p < 0.0001 |
- Note: Minimum time points were reported as the first timepoint in which the life stage was observed in a sample.
| Stage | 24°C | 37°C | Statistical significance |
|---|---|---|---|
| First instar | 62.67 ± 4.39 | 28.94 ± 1.03 | F = 55.86; df = 1, 17; p < 0.0001 |
| Second instar | 104.00 ± 4.91 | 45.83 ± 1.66 | F = 126.12; df = 1, 17; p < 0.0001 |
| Third instar | 165.11 ± 2.97 | 73.39 ± 1.59 | F = 739.95; df = 1, 17; p < 0.0001 |
| Post-feeding third instar | 183.11 ± 5.41 | 95.61 ± 8.71 | F = 72.89; df = 1, 15; p < 0.0001 |
- Note: Maximum time points reported as the timepoint following the last sample in which the life stage was observed. Maximum data were not recorded for pupae nor was adult longevity measured in this study.
2.5 Time of colonization estimations
Time of colonization (TOC) estimations were calculated using the standard accumulated degree day model accepted in the discipline. This was completed to highlight how differences in published datasets may result in different time of colonization estimations when used by practitioners in applied scenarios. Degree days were calculated using the formula (((rearing temperature − lower development threshold)*mean hours to reach life stage)/24). A lower development threshold (LDT) temperature of 12°C was used [25]. Accumulated degree day (ADD) units were used rather than accumulated degree hour (ADH) units because oftentimes when development data are used in casework, it must be converted to ADD units due to the weather data being reported in daily averages rather than hourly averages.
2.6 Statistics
All statistical analyses were performed using JMP Pro 16 [30]. An ANOVA was run to determine whether significant differences existed between temperatures within a developmental stage. The assumptions of normality were tested using a Shapiro–Wilk test, and equal variances were tested using a Bartlett's test. All assumptions of an analysis of variance (ANOVA) were met unless otherwise stated. For all tests, an alpha of 0.05 was used.
3 RESULTS
3.1 Minimum and maximum development rates
The duration of development was significantly (p < 0.05) different across temperatures for all stages (Tables 1 and 2). Larvae reared at 37°C developed c. 52% faster than larvae reared at 24°C across all life stages with reduced variation in development times when assessing average minimum time to reach each life stage (Table 1). Larvae reared at 37°C developed c. 53% faster than larvae reared at 24°C across all life stages with reduced variation in development times when assessing average maximum time to reach each life stage (Table 2).
3.2 Calculated data for comparison with Wang et al. (2018)
Accumulated degree days (ADD) were calculated for this study as well as for Wang et al. [25], the only existing dataset available for use by forensic entomology practitioners for time of colonization estimations, using the closest developmental temperatures to those used in this study. Comparisons of the raw and calculated data as they would be used by forensic practitioners for both studies are presented in Table 3.
| Stage | Raw hours, current study (24°C) | ADDa, current study (24°C) | Raw hours, Wang et al. (25°C) | ADDa, Wang et al. (25°C) | Raw hours, current study (37°C) | ADDa, current study (37°C) | Raw hours, Wang et al. (34°C) | ADDa, Wang et al. (34°C) |
|---|---|---|---|---|---|---|---|---|
| First instar | 18.67 | 9.34 | 18.50 | 10.02 | 9.06 | 9.43 | 10.50 | 9.63 |
| Second instar | 53.78 | 26.89 | 44.70 | 24.21 | 25.61 | 26.68 | 24.00 | 22 |
| Third instar | 91.56 | 45.78 | 73.20 | 39.65 | 40.50 | 42.19 | 40.30 | 36.94 |
| Post-feeding third instar | 158.57 | 79.29 | NA | NA | 72.94 | 75.98 | NA | NA |
| Pupa | 176.89 | 88.45 | 140.40 | 76.05 | 84.06 | 87.56 | 81.50 | 74.71 |
| Adult | 334.44 | 167.22 | 297.40 | 161.09 | 172.06 | 179.22 | 155.50 | 142.54 |
- Abbreviation: NA, not available due to reporting of data in original study.
- a A lower development threshold (LDT) of 12°C was used for degree day calculations.
3.3 Comparisons of time of colonization estimations using both studies
ADD using the previously mentioned LDT for both this study and Wang et al. [25] were used to provide TOC estimations for constant environmental temperatures averaging 37°C (Table 4) and 24°C (Table 5). This was to represent theoretical casework calculations for cases of human myiasis and remains discovered indoors at an approximate room temperature in Texas, USA, respectively. TOC estimations derived using Wang et al. [25] were consistently shorter for most life stages than those derived using this study at the higher theoretical environmental temperature (37°C). At the lower theoretical environmental temperature (24°C), TOCs from this study were in agreement with TOCs derived from Wang et al. (2018). In their study, Wang et al. [25] combined the duration of feeding and post-feeding stages of the third instar, therefore as presented in Tables 4 and 5, those TOC estimations are the same for that study.
| Development stage of evidence collected | Current study (37°C) TOC range (day) | Wang et al. (34°C) TOC range (day) |
|---|---|---|
| Egg | <1 | <1 |
| First instar | <1 to 2 | <1 to 1 |
| Second instar | 2 | 1 to 2 |
| Feeding third instar | 2 to 4 | 2 to 4 |
| Post-feeding third instar | 4 | 2 to 4 |
| Pupa (i.e., intact pupa) | 4 to 8 | 4 to 6 |
| Puparium (i.e., eclosed pupa) | 8+ | 6+ |
| Development stage of evidence collected | Current study (24°C) TOC range (day) | Wang et al. (25°C) TOC range (day) |
|---|---|---|
| Egg | <1 | <1 |
| First instar | <1 to 3 | <1 to 3 |
| Second instar | 3 to 4 | 3 to 4 |
| Feeding third instar | 4 to 7 | 4 to 7 |
| Post-feeding third instar | 7 to 8 | 4 to 7 |
| Pupa (i.e., intact pupa) | 8 to 14 | 7 to 14 |
| Puparium (i.e., eclosed pupa) | 14+ | 14+ |
4 DISCUSSION
Forensic entomologists rely on development data to determine time of colonization of living or deceased individuals by arthropods. Due to such importance, numerous life-history studies have been published across a diverse group of dipteran species [31-33]. Furthermore, research has determined that a number of factors, biotic and abiotic, can impact the development patterns associated with a given species. Biotic criteria, such as density [34], genetics [35], intraspecific competition, and interspecific competition [36], can accelerate, or suppress, development depending on circumstances at play. The same can be said for abiotic factors, such as temperature [37], humidity, and tissue type/moisture [38].
Thus, given the relevance of the house fly to forensics, studies examining the impact of such factors on development are essential. Having such data allow for insights (e.g., time of colonization) that might be challenging to determine with methods other than entomology or when attempting to apply datasets from other locations. Furthermore, creating baseline development data serves as the cornerstone for determining the impact of these factors on house fly progression through its life cycle. In the end, by determining such influences, applications of said data could lead to greater precision and accuracy when determining time of colonization.
As demonstrated in this study, temperature significantly impacts the development of the house fly, as a shift in temperature alone from 24°C to 37°C can accelerate development by 48.5%. This outcome is not surprising given its dependence on external heat to develop. For example, blow flies, flesh flies, and filth flies/house flies are often the key taxa collected from vertebrate remains [39-41], but the primary families and species found are dependent on geographical location, elevation, and circumstances [42-45]. In most vertebrate decomposition events, blow flies are the most common family found. However, in cases of abuse/neglect, when fecal matter or similar substances may be present on a living person or in their immediate surroundings, filth flies, such as the house fly, can be the primary or sole colonizer (personal casework). With the species that have been studied across Calliphoridae, Sarcophagidae, and Muscidae, temperature has been determined to be a key factor driving the rate of development [13]. In the case of the house fly, similar results to those in this study were determined for a population out of China [25]. To the knowledge of the authors, at the time of this publication, Wang et al.'s [25] study is the only other known study focusing on the development rate of this species in a forensic context with development time reported for all individual development stages.
Tables 4 and 5 provide examples of the utilization of data from Wang et al. [25] and the current study to demonstrate the difference in development rates generated by both studies and how this could impact time of colonization estimations derived by using each study. There are key differences in the study methodologies and data recording, which may contribute to the resulting differences in the resulting applications of data. Wang et al. [25] used a M. domestica population collected from Suzhou, China, whereas the current study used a population from Stephenville, TX, USA. Geographically distinct populations of the same species have been shown to have significantly different development rates in other dipteran species; Owings et al. [35] found that three populations of Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae) separated by c. 240 km radius exhibited variation in their development rates, with one population developing between ~6% and 13% faster under varying conditions. Rearing conditions also differed; Wang et al. [25] reared larvae at 34°C and 75% RH with a 12:12 L:D cycle, while the current study utilized developmental conditions of 37°C and 70% RH with a 14:10 L:D cycle. The temperature in the current study was used as it approximates average body temperature of a living person to increase its applicability to abuse or neglect casework. Additionally, Wang et al. [25] used a mixture of fresh lean pork and wheat bran for a rearing diet, while the current study used whole (i.e., not ground or minced) lean pork to correlate with human tissue. Tissue type can have a significant effect on development rate; Thyssen et al. [46] found a significant (p < 0.0001) difference in development time for Chrysomya albiceps Wiedemann, Chrysomya megacephala Fabricius, and Chrysomya putoria Wiedemann (Diptera: Calliphoridae) reared on bovine tongue, bovine stomach, bovine muscle, chicken heart, and bovine liver tissue. The final difference worth noting when assessing the differences in Tables 4 and 5 are small but significant differences in the recording and reporting of data for individual developmental stages. Wang et al. [25] reported a development time for the third instar as a whole (i.e., completion of second instar to onset of pupation), whereas the current study separated the third instar into the feeding and post-feeding (i.e., wandering) third-instar stages. The third instar for the house fly, much like other dipterans of forensic importance, requires the largest amount of time to complete compared with other larval stages (see Flores et al. [11] and Boatright and Tomberlin [31] for examples, where the feeding third instar accounts for up to 35% of total development time at certain conditions). By partitioning this stage into two portions and reporting data in this manner, more precise time of colonization estimates can be made when third instar M. domestica are present.
There is presently no published recommendations or best practices for development study publications in forensic entomology, and thus, differences in study methodology are seen often and can have direct effects on the resulting application of the data in casework. While the sampling method used in the current study (subsampling without replacement) is the most common experimental method used in development data studies [9-11, 31, 32, 36, 47-53], there is an inherent limitation in that not all individuals are being assessed at each time point. Therefore, there is the potential that an earlier or later life stage may be missed during any given sampling time check. However, an unsystematic method was used to sample larvae in this study, meaning neither the largest nor the smallest larvae were targeted as that sampling method has been shown to yield models very similar to those of complete datasets [47]. Three larvae were sampled at each time point every 4–6 h (a total of 12–18 larvae were sampled within a 24 h time frame from each larval container) from each replicate container in each temperature treatment in each trial. The authors acknowledge that sampling more larvae would inherently increase the confidence [54]; the authors chose to prioritize other physiological aspects of paramount importance to dipteran larval development, including but not limited to intentionally not over-sampling to allow for an increased number of larvae to remain in each replicate container to allow for increased group exodigestion as seen in maggot masses in real-world scenarios.
The current study provides the first report of development rates of M. domestica for a population in the United States at forensically relevant temperatures. Musca domestica is routinely collected in a wide range of casework ranging from cases of suspected abuse and neglect to investigations of death, both natural and criminal [23, 25-27] (CF, JRC, JKT, unpublished data). The lack of existing development data available assessed at relevant temperatures and reported in a functional manner for use by a practicing forensic entomologist has potentially limited the scope of information these practitioners have historically been able to provide when analyzing evidence that includes this species. This study fills a key gap in forensic entomology literature that can be of use immediately to practicing professionals in the field.
CONFLICT OF INTEREST STATEMENT
The authors have no conflicts of interest to report.
