DIRECT INJURY,,FORENSICS Effects of Temperature and Tissue Type on the Development of macellaria (Diptera: )

1 1,2 STACY A. BOATRIGHT AND JEFFERY K. TOMBERLIN

J. Med. Entomol. 47(5): 917Ð923 (2010); DOI: 10.1603/ME09206 ABSTRACT The secondary screwworm, (Fabricius), was reared on either equine gluteus muscle or porcine loin muscle at 20.8ЊC, 24.3ЊC, and 28.2ЊC. C. macellaria needed Ϸ35% more time to complete development when reared at 20.8 than 28.2ЊC. Furthermore, larval growth and weight over time did not differ between larvae reared on equine versus porcine muscle. This study is the second in the United States to examine the development of C. macellaria and is the Þrst to examine development of this species on muscle tissue from different vertebrate species. These data could provide signiÞcant information regarding time of colonization, including myiasis and neglect cases involving humans and . Furthermore, these results in comparison with the only other data set available for this species in North America indicate a fair amount of phenotypic variability as it relates to geographic location, suggesting caution should be taken when applying these data to forensic cases outside the region where this study was conducted.

KEY WORDS secondary screwworm, , forensic veterinary medicine, myiasis

Forensic entomology is the utilization of and though similar development times have been docu- other as evidence in both civil and criminal mented in pre-existing data sets for a variety of blow investigations (Williams and Villet 2006). The broad ßy species, some variations can be found within spe- scope of this Þeld can be broken down into three cies (Tarone and Foran 2006, Gallagher et al. 2010). subdivisions, which are urban, stored-product, and Variation could be the result of a number of factors, medicolegal entomology (Archer 2007). Medicolegal such as experimental design, environment, and ge- entomology relates to necrophagous arthropods col- netic variation. Tarone and Foran (2006) determined onizing and feeding on living and necrotic human and that environment conditions greatly inßuenced the tissue. Insects found on vertebrate remains development of the blow ßy sericata (Meigen) may be used to estimate the period of activity (Diptera: Calliphoridae). Their Þndings demonstrate or minimum postmortem interval (Amendt et al. the need for multiple development data sets for each 2007). species of blow ßy species across geographic regions, Entomologists also are asked to analyze insect ev- as well as for a standardization of laboratory-rearing idence collected from living people and animals. Con- techniques. Þrming myiasis, the infestation and subsequent feed- Most development data used by forensic entomol- ing upon living tissue, organs, or bodily ßuids of a ogists are obtained from studies examining the growth vertebrate host by dipteran larvae can be used as of larvae-fed beef liver (Clark et al. 2006). However, evidence in cases of suspected neglect or abuse it has been demonstrated that the host species on (Anderson and Huitson 2004). Myiasis typically oc- curs in elderly or very young people, who are unable which the larvae feed may signiÞcantly alter larval to care for themselves, as well as in pets and livestock growth rate. For example, development of L. sericata- with open wounds or fecal matter present on their fed lung, liver, and heart, from both cows and swine, bodies for an extended period of time (Anderson and was compared (Clark et al. 2006). It was determined Huitson 2004). that larvae grew signiÞcantly faster on swine than on Forensic entomologists depend on growth data cow. Furthermore, development varied when reared from laboratory studies to estimate the period of insect on lung and heart compared with liver of the same activity for blow ßies (Diptera: Calliphoridae) col- animal. For forensic entomologists, such results em- lected from decedents (Tarone and Foran 2006) as phasize the need for development data sets across well as the living (Anderson and Huitson 2004). Al- tissue types as well as across species. The importance of recording the location from which blow ßy larvae 1 Department of Entomology, Texas A&M University, College Sta- are removed from a corpse at a body recovery scene tion, TX 77843. was also demonstrated (Clark et al. 2006). Such in- 2 Corresponding author: 2475 TAMU, Department of Entomol- ogy, Texas A&M University, College Station, TX 77843 (e-mail: formation can allow for reÞned estimates of the period [email protected]). of insect activity.

0022-2585/10/0917Ð0923$04.00/0 ᭧ 2010 Entomological Society of America 918 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 47, no. 5

Cochliomyia macellaria (Fabricius), the secondary were partitioned into 200-g samples and placed into screwworm (Diptera: Calliphoridae), is a facultative separate Ziploc sandwich bags, labeled, assigned a parasite. Unlike the primary screwworm, Cochliomyia temperature treatment, and stored in a Ϫ20ЊC freezer hominivorax (Coquerel) (Diptera: Calliphoridae), until use. larvae of C. macellaria do not feed on living tissue; Experiment Design. The experimental design was rather, they colonize a wound and feed on necrotic adapted from Byrd and Butler (1996) and Nabity et al. tissue (Harrison and Pearson 1968). C. macellaria is (2006). Development time of C. macellaria on equine one of the most common colonizers of vertebrate and porcine muscle tissue in 136LLVL Percival up- remains in the southern United States, thus making it right incubators set at 20.8 Ϯ 1.3ЊC, 24.3 Ϯ 0.7ЊC, and a ßy of great forensic importance (Hall 1948). The 28.2 Ϯ 1.1ЊC was recorded. An Onset HOBO U12Ð006 secondary screwworm is of particular importance to data logger with an Onset TMC6-HD air, water, and the livestock industry because of large economic soil temperature sensor (Onset, Pocasset, MA) was losses resulting from pathogen transmission and my- placed on each shelf in each of the growth chambers, iasis (Anderson and Huitson 2004). The recent and temperature was recorded hourly throughout the increase in the number of criminal and civil investi- duration of the study. gations in the United States where forensic entomol- Equine and pork tissues were removed from the ogists are asked to analyze evidence further demon- freezer, and 200-g samples were placed individually in strates the need for more development data sets to 1.10-liter styrene mosquito-breeding containers (Bio- enhance the accuracy of estimating the period of in- Quip Products, Rancho Dominguez, CA). These were sect activity (Grassberger and Reiter 2001). Cur- held individually in opaque Sterilite shoebox contain- rently, the only available development data for C. ers (Sterilite, Townsend, WI), 33 (L) ϫ 19 (W) ϫ 11 macellaria from the United States are for specimens (D) cm, layered with 500 ml of sand (Quikrete Pre- reared on lean pork in Florida (Byrd and Butler 1996). mium Play Sand, Atlanta, GA) to prevent wandering The primary objective of this study was to examine larvae from escaping. The containers were not cov- the development of C. macellaria on equine and por- ered and were placed in a Latin square design with cine striated muscle tissue at different temperatures. three samples of each tissue type in each growth We hypothesized that the development rates for C. chamber. Tissue samples were inoculated with macellaria would not differ across tissue types. Fur- Ϸ24 h after placement in the growth chambers. thermore, we hypothesized that C. macellaria devel- colonies were monitored hourly for oviposition. Eggs opment would not differ across the temperatures in- Ͻ1 h old were mixed in deionized water and weighed vestigated. gravimetrically, and 400 aliquots were placed on each tissue sample, using a Þne-tip artist paint brush. Only eggs from 7- to 10-d-old F generation ßies were Materials and Methods 1 used in this study. Adult Fly Colony. C. macellaria used in this exper- Life History. Eggs on each tissue sample were iment originated from larvae collected from decom- checked hourly for larval eclosion. Once initial eclo- posing animal carcasses in the vicinity of College Sta- sion occurred for eggs on a tissue sample, observations tion, TX, from June through August 2008. Collected were made every 12 h. During each observation, three larvae were fed fresh beef liver in a 1-liter plastic larvae were subsampled from the respective con- container and housed in a 136LLVL Percival (Percival tainer. Each sampled was parboiled in water, and ScientiÞc, Perry, IA) growth chamber at Ϸ27ЊC with then weight, length, , and time of observation 14:10 (L:D) h and 75Ð80% RH (modiÞed from Byrd were recorded. Weights were measured using an Ad- and Butler 1996, Nabity et al. 2006). Emergent adult venture-Pro AV64 Ohaus scale (Ohaus, Pine Brook, ßies were placed in nylon mesh 30 ϫ 30 ϫ 30-cm NJ). Lengths of larvae were recorded using a stage Bioquip cages and provided a 50:50 sugar:powdered scale and a Meiji Techno EMZ-8TR microscope (Meiji milk mixture and fresh water ad libitum (Byrd and Techno America, Santa Clara, CA). These observa- Butler 1996). tions continued until all larvae had either died or Tissue Source. Tissue samples from three equines, pupated. Equus ferus Boddaert, and three porcines, Sus scrofa L., Development time of pupae was recorded for the were used in this development study. Equine gluteus initial 30 larvae to pupate. Pupae were placed indi- muscle was supplied by the Texas Veterinary Medical vidually in 35-ml Jetware (Jetware, HatÞeld, PA) plas- Diagnostic Laboratory (Texas A&M University, Col- tic medicine cups containing Ϸ10 ml of sand lege Station, TX). Tissue acquisition protocols were (Quikrete Premium Play Sand, Atlanta, GA), capped approved by the Animal Welfare Assurance Program, with breathable lids, labeled, returned to the appro- Texas A&M University. Equines used were free of priate growth chamber, and monitored every 12 h for barbiturates or other materials used for euthanization. adult emergence. Time to adult emergence and sex Approximately 4 kg of tissue was collected from each were recorded for each individual. Sex of each ßy was of the three equines. Lean boneless pork chops (i.e., recorded after death. Adults that died during eclosion loin muscle) were purchased from a local grocery. were not included in adult longevity analysis. Because Varied packets of boneless pork chops were purchased of unequal subsample sizes per sample, development to increase the likelihood of the tissue resulting from data were not analyzed by sex; however, overall sex different animals. Equine and porcine tissue samples ratio was determined. Adults were monitored every September 2010 BOATRIGHT AND TOMBERLIN:DEVELOPMENT OF SECONDARY SCREWWORM 919

Fig. 1. C. macellaria larval weight (n ϭ 3) Ϯ SE developing at three temperatures over time.

12 h for mortality. Adult ßies were provided with 0.20 development did not differ across tissue treatments. ml of distilled water via a 1-ml Kendall Monoject Larval weight did not signiÞcantly differ between tis- SoftPack insulin syringe (Kendall, MansÞeld, MA) in- sue types at 20.8ЊC(F ϭ 0.157; df ϭ 1, 65; P ϭ 0.693), serted through the lid every 24 h. Development and 24.3ЊC(F ϭ 1.005; df ϭ 1, 54; P ϭ 0.321), or 28.2ЊC(F ϭ longevity times were converted to degree days with 1.380; df ϭ 1, 41; P ϭ 0.247) (Fig. 3). Hours elapsed the base temperature set at 10ЊC. Representative sam- signiÞcantly predicted larval weight at 20.8ЊC (slope ϭ ples of the adult specimens obtained from this study 0.993; t ϭ 0.993; P Ͻ 0.0001), 24.3ЊC (slope ϭ 1.214; were deposited in the Texas A&M University Insect t ϭ 12.224; P Ͻ 0.0001), and 28.2ЊC (slope ϭ 1.708; t ϭ Collection. 12.805; P Ͻ 0.0001). Hours elapsed also explained a Statistical Analysis. All statistical analyses were per- signiÞcant proportion of variance in ranked larval formed using SPSS (SPSS 2005). Analysis of covari- weight for 20.8ЊC(R2 ϭ 0.875; df ϭ 3, 65; P Ͻ 0.0001), ance (ANCOVA) was used to compare weight and 24.3ЊC(R2 ϭ 0.863; df ϭ 3, 54; P Ͻ 0.0001), and 28.2ЊC length (dependent variables) data for ßies reared on (R2 ϭ 0.880; df ϭ 3, 41; P Ͻ 0.0001). Hours elapsed each tissue type across temperatures (independent signiÞcantly predicted larval length at 20.8ЊC (slope ϭ variables) over time (covariate). Because weight and 1.020; t ϭ 14.529; P Ͻ 0.0001), 24.3ЊC (slope ϭ 1.225; length data did not meet basic assumptions for para- t ϭ 11.808; P Ͻ 0.0001), and 28.2ЊC (slope ϭ 1.735; t ϭ metric analysis, they were ranked before analysis. De- 10.990; P Ͻ 0.0001) (Fig. 4). Hours elapsed also ex- velopment times for stage of development were con- plained a signiÞcant proportion of variance in ranked verted to accumulated degree days and analyzed with larval length for 20.8ЊC(R2 ϭ 0.868; df ϭ 3, 65; P Ͻ a two-way analysis of variance, with temperature and 0.0001), 24.3ЊC(R2 ϭ 0.850; df ϭ 3, 54; P Ͻ 0.0001), and tissue as main effects and samples nested within tem- 28.2ЊC(R2 ϭ 0.838; df ϭ 3, 41; P Ͻ 0.0001). perature. Fisher least signiÞcant difference test was Intercept for larval length by hour did not signiÞ- performed to separate means following a signiÞcant F cantly differ between tissue types at 20.8ЊC(F ϭ 0.505; test (P Ͻ 0.05). df ϭ 1, 65; P ϭ 0.480), 24.3ЊC(F ϭ 0.742; df ϭ 1, 54; P ϭ 0.393), or 28.2ЊC(F ϭ 0.912; df ϭ 1, 41; P ϭ 0.345) (Fig. 4). Therefore, tissue was removed from the analysis, Results and ranked mean data for weight and length were Nontransformed mean length and weight data over analyzed with an ANCOVA, with temperature as the time for larvae reared on equine and porcine tissue are independent variable and hour as the covariate. How- presented in Figs. 1 and 2, respectively. Based on our ever, temperature and hour interacted for length (F ϭ analyses, we failed to reject our null hypothesis. Larval 16.03; df ϭ 2, 166; P Ͻ 0.0001) and weight (F ϭ 16.19;

Fig. 2. C. macellaria larval length (n ϭ 3) Ϯ SE developing at three temperatures over time. 920 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 47, no. 5

Fig. 4. Ranked length (cm) data using the calculated y-intercept and ANCOVA results for larvae reared at (a) 20.8ЊC, (b) 24.3ЊC, and (c) 28.2ЊC.

ture and instar was also determined (F ϭ 21.636; df ϭ Fig. 3. Ranked weight (g) data using the calculated x- 4, 4; P ϭ 0.0006) in regard to degree days needed to intercept and ANCOVA results for larvae reared at (a) complete each stage of development. reared at 20.8ЊC, (b) 24.3ЊC, and (c) 28.2ЊC. 28.2ЊC needed Ϸ31 and 21% less degree days to com- plete development than those reared at 20.8ЊC and 24.3ЊC, respectively. Sex ratios were similar (Table 2) df ϭ 2, 166; P Ͻ 0.0001). Therefore, the results of the across tissue and temperature. Although not signiÞ- ANCOVA could not be used. cant, adult longevity (Table 2) was similar across tis- Degree day data for C. macellaria reared on equine sue treatments, with those reared at cooler tempera- and porcine tissue are presented in Table 1 using a tures living longer. base temperature of 10ЊC. Based on the analysis of variance results, we failed to reject our null hypoth- Discussion esis. Degree days needed for adults reared on porcine and equine muscle were not signiÞcantly different Byrd and Butler (1996) assessed the development of (F ϭ 1.540; df ϭ 1, 2.5; P ϭ 0.318) (Table 1), indicating C. macellaria on porcine tissue across Þve temperature no tissue effect. However, we did reject our null hy- regimes. Their study included temperatures of 21.1ЊC pothesis that C. macellaria development would not and 26.7ЊC, which should have produced mean devel- signiÞcantly differ across the temperatures investi- opment stage values comparable to those recorded at gated. Degree days by instar did in fact differ signif- 20.8ЊC and 28.2ЊC in our study. However, ßies in our icantly (P Ͻ 0.05). An interaction between tempera- study required more degree days to complete each September 2010 BOATRIGHT AND TOMBERLIN:DEVELOPMENT OF SECONDARY SCREWWORM 921 on equine (3 ؍ Table 1. Minimum degree days and accumulated degree days for development stages of Cochliomyia macellaria (n and porcine muscle tissue at three temperatures

Second Temperature Egg Ϯ SE First instar Ϯ Third instar Ϯ Prepupal Ϯ Pupal Ϯ SE Tissue instar Ϯ SE (ЊC) (DD) SE (DD) SE (DD) SE (DD) (DD) (DD) 20.8 Equine 8.32 Ϯ 0.39 10.90 Ϯ 0.00 30.87 Ϯ 3.64 59.92 Ϯ 6.29 8.60 Ϯ 0.55 69.89 Ϯ 3.12 Mean ADD 19.22 50.09 110.01 118.61 188.50 Porcine 8.02 Ϯ 0.31 10.90 Ϯ 0.00 30.87 Ϯ 3.64 65.37 Ϯ 3.15 7.15 Ϯ 0.60 66.17 Ϯ 0.49 Mean ADD 18.92 49.79 115.16 122.31 188.48 24.3 Equine 10.82 Ϯ 0.39 14.18 Ϯ 0.00 37.80 Ϯ 2.36 66.15 Ϯ 6.25 9.57 Ϯ 1.83a 64.82 Ϯ 1.73 Mean ADD 25.00 62.80 128.95 138.52 203.34 Porcine 10.24 Ϯ 0.19 14.18 Ϯ 0.00 33.08 Ϯ 2.36 70.88 Ϯ 0.00 8.62 Ϯ 0.47 62.63 Ϯ 0.96 Mean ADD 24.42 57.50 128.38 137.00 199.63 28.2 Equine 10.11 Ϯ 0.25 14.80 Ϯ 2.96 26.63 Ϯ 0.00 62.14 Ϯ 5.13 11.10 Ϯ 1.82a 64.37 Ϯ 3.39 Mean ADD 24.91 51.54 113.68 124.78 189.15 Porcine 9.62 Ϯ 0.43 14.80 Ϯ 2.96 32.56 Ϯ 2.96 71.02 Ϯ 5.13 10.26 Ϯ 0.29 64.97 Ϯ 1.15 Mean ADD 24.42 56.98 128.00 138.26 203.23

Base temperature ϭ 10ЊC. DD, degree day; ADD, accumulated degree days. a n ϭ 2. stage of development than those in Byrd and Butler important to note that these studies (Greenberg 1991, (1996). The only exceptions to this observation were Byrd and Butler 1996, Byrd and Allen 2001) compared for the egg stage at 28.2ЊC and the Þrst instar stage at development for a single population of the target spe- 20.8ЊC, both of which took less degree days to com- cies within a given region. Our comparison with Byrd plete development in our study. and Butler (1996) represents two unique geographic One factor that might explain the difference be- regions and potentially unique populations of C. ma- tween the studies is that Byrd and Butler (1996) used cellaria (Gallagher et al. 2010). oscillating temperature regimes for their 21.1ЊC and Although largely untested, genetic variability may 26.7ЊC treatments, each with an amplitude of 5.5ЊC, to be an important source of development variability simulate naturally occurring conditions in Florida. when comparing different populations (Tarone and Temperatures in our study were relatively stable. Foran 2006). Several authors have generated devel- Behrens et al. (1983) determined that development opment data for different populations of L. sericata of the cricket, Gryllus bimaculatus, De Geer (Or- (Kamal 1958, Greenberg 1991, Anderson 2000, Grass- thoptera: Gryllidae), under oscillating temperature berger and Reiter 2001). Each of these populations is Ϸ conditions was 26% faster than when reared under from a unique ecological setting, and the resulting constant temperature conditions; however, it should development degree days differed. Kamal (1958), be noted that in our study the mean temperatures and Greenberg (1991), and Grassberger and Reiter (2001) larval food taxon were different than those in Byrd and estimated faster minimum stage durations than Butler (1996). In contrast, and more applicable to our Anderson (2000). Ames and Turner (2003) deter- study, Clarkson et al. (2004) determined that ßuctu- mined (Robineau-Desvoidy) (Diptera: ating versus constant temperatures delayed develop- Calliphoridae) and (Linnaeus) ment of larvae of Protophormia terraenovae (Rob- (Diptera: Calliphoridae) from different geographic lo- ineau-Desvoidy) (Diptera: Calliphoridae). Others, cations have different thermal constant accumulated de- including Greenberg (1991), Byrd and Allen (2001), gree hour values. and as discussed throughout Byrd and Butler (1996), A third factor explaining the variation observed determined that development of blow ßies reared between our study and Byrd and Butler (1996) is under ßuctuating temperatures was retarded in com- differences in laboratory protocols for rearing C. ma- parison with those reared under constant conditions, which is in contrast to our Þndings. However, it is cellaria. Such factors include the difference in han- dling methods during rearing, as well as the use of different rearing chambers. Consequently, it is impor- Table 2. Sex ratio, degree days for longevity of male and tant to standardize rearing conditions, which best on equine and porcine (3 ؍ female Cochliomyia macellaria (n muscle tissue at three temperatures mimic growth on actual to compare blow ßy data sets and use them in legal investigations in dif- Temperature Sex % ratio Longevity (DD) ferent regions of the country (Tarone and Foran Tissue (ЊC) ((:⅙) ( Ϯ SE & Ϯ SE 2006). If such standardized protocols were developed 20.8 Equine 44.3:55.7 39.9 Ϯ 1.7 39.9 Ϯ 1.2 by forensic entomologists, researchers could then fo- Porcine 52.1:47.9 43.6 Ϯ 0.2 43.3 Ϯ 0.5 cus their work toward understanding the develop- 24.3 Equine 52.9:47.1 26.2 Ϯ 4.7 31.2 Ϯ 2.9 ment differences between populations that can be Porcine 53.6:46.4 35.7 Ϯ 2.2 36.2 Ϯ 3.7 attributed to genetic variability. 28.2 Equine 33.9:66.1 22.9 Ϯ 0.5 22.2 Ϯ 0.7 Porcine 50.4:49.6 26.4 Ϯ 1.3 26.8 Ϯ 0.6 Our study assessed the development of C. macel- laria on equine and porcine striated muscle tissue. Base temperature ϭ 10ЊC. DD, degree day. Although we observed no signiÞcant difference in 922 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 47, no. 5 mean larval length or weight for specimens reared on References Cited equine and porcine muscle, Clark et al. (2006) deter- Amendt, J., C. P. Campobasso, E. Gaudry, C. Reiter, H. N. mined in their study that the species origin on which LeBlanc, and M.J.R. Hall. 2007. Best practice in forensic L. sericata larvae were reared indeed inßuenced entomology: standards and guidelines. Int. J. Legal Med. weight and length over time. However, Clark et al. 121: 90Ð104. (2006) determined that tissue source did have an ef- Ames, C., and B. Turner. 2003. Low temperature epi- fect on development time, which is similar to our sodes in development of blowßies: implications for results. They determined that L. sericata needed Ϸ31 postmortem interval estimation. Med. Vet. Entomol. 17: h additional to complete the feeding stage and were 2 178Ð186. Anderson, G. S. 2000. Minimum and maximum develop- mm shorter when fed porcine liver rather than lung. ment rates of some forensically important Calliphoridae Similarly, C. vicina fed porcine lung, heart, kidney, or (Diptera). J. Forensic Sci. 45: 824Ð832. brain completed the feeding stages 2 d sooner than Anderson, G. S., and N. R. Huitson. 2004. Myiasis in pet those fed porcine liver (Kaneshrajah and Turner animals in British Columbia: the potential of forensic 2004). Consequently, until we have a better under- entomology for determining duration of possible neglect. standing of the variation in larval development as it Can. Vet. J. 45: 993Ð998. relates to tissue fed upon, we recommend that such Archer, M. 2007. Forensic entomology. Australian Police J. 61: 66Ð70. data sets be applied in a conservative manner when Behrens, W., K. H. Hoffman, S. Kempa, S. Ga¨ßler, and G. analyzing larval evidence collected from a living or Merkel-Wallner. 1983. Effects of diurnal thermoperiods deceased individual. Otherwise, generalizing larval and quickly oscillating temperatures on the development development regardless of the tissue fed could result and reproduction of crickets, Gryllus bimaculatus. Oeco- in an over- or underestimate of the larval age and, logia 59: 279Ð287. consequently, the period of insect activity. Byrd, J. H., and J. C. Allen. 2001. The development of the Most research on the development of forensically black blow ßy, Phormia regina (Meigen). Forensic Sci. important blow ßies has focused on gathering data for Int. 120: 79Ð88. Byrd, J. H., and J. F. Butler. 1996. Effects of temperature on the egg, larval, and pupal stages (Greenberg 1991, Cochliomyia macellaria (Diptera: Calliphoridae) devel- Grassberger and Reiter 2001, Tarone and Foran 2006). opment. J. Med. Entomol. 33: 901Ð905. Although some data on adult longevity for such blow Clark, K., L. Evans, and R. Wall. 2006. Growth rates of the ßies do exist (Gabre et al. 2005), our study is the Þrst blowßy, Lucilia sericata on different body tissues. Foren- to break down adult longevity based on sex (Table 2). sic Sci. Int. 156: 145Ð149. Our results indicate that males and females each live Clarkson, C. A., N. R. Hobischak, and G. S. Anderson. 2004. for comparable amounts of degree days after emer- A comparison of the development rate of Protophormia terraenovae (Robineau-Desvoidy) raised under constant gence. This information could be useful in abuse or and ßuctuating temperature regimes. Can. Soc. Forensic death investigations in which empty pupal cases and Sci. 37: 95Ð101. dead adults are collected from indoor body recovery Gabre, R. M., F. K. Adham, and H. Chi. 2005. Life table of sites. This information could provide a conservative megacephala (Fabricius) (Diptera: Callipho- estimate of the time since emergence and, conse- ridae). Acta Oecologica 27: 179Ð183. quently, a more reÞned estimate of the period of insect Gallagher, M. B., S. Sandhu, and R. Kimsey. 2010. Variation activity using the degree day estimates. in development time for geographically distinct popula- tions of the common green bottle ßy, Lucilia sericata Our study produced the second development data (Meigen). J. Forensic Sci. 55: 438Ð442. set for C. macellaria in the United States. These data Grassberger, M., and C. Reiter. 2001. Effect of temperature offer new insight to variation in development across on Lucilia sericata (Diptera: Calliphoridae) development tissue types and temperatures as related to accu- with special reference to the isomegalen- and isomor- mulated degree days, larval length, and weight. In phen-diagram. Forensic Sci. Int. 120: 32Ð26. addition, these data could be used to determine the Greenberg, B. 1991. Flies as forensic indicators. J. Med. En- period of insect activity for C. macellaria specimens tomol. 28: 565Ð577. Hall, D. G. 1948. The Blowßies of North America. Thomas collected from human, as well as livestock, remains Say Foundation, Baltimore, MD. in Texas. However, based on development differ- Harrison, B. A., and W. G. Pearson. 1968. A case of aural ences observed between our study and Byrd and myiasis caused by Cochliomyia macellaria (Fabricius). Butler (1996), additional studies are needed for Military Med. 133: 484Ð488. other regions of the United States to gain a better Kamal, A. S. 1958. Comparative study of thirteen species of understanding of this variation and its application in sarcosaprophagous Calliphoridae and Sarcophagidae forensic investigations. (Diptera). I. Bionomics. Ann. Entomol. Soc. Am. 51: 261Ð 271. Kaneshrajah, G., and B. Turner. 2004. Calliphora vicina larvae grow at different rates on different body tissues. Acknowledgments Int. J. Legal Med. 118: 242Ð244. Nabity, P. D., L. G. Higley, and T. M. Heng-Moss. 2006. We thank K. Schoenly, J. Cammack, H. LeBlanc, A. Effects of temperature on development of Phormia regina Tarone, J. Byrd, R. Mohr, and three anonymous reviewers for (Diptera: Calliphoridae) and use of development data in their time and effort reviewing earlier versions of this manu- determining time intervals in forensic entomology. script. J. Med. Entomol. 43: 1276Ð1286. 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SPSS. 2005. SPSS for Windows, release 14.0.1 computer pro- Williams, K. A., and M. H. Villet. 2006. A history of southern gram, version 14.0. SPSS, Chicago, IL. African research relevant to forensic entomology. S. Af- Tarone, A. M., and D. R. Foran. 2006. Components of de- rican J. Sci. 102: 59Ð65. velopmental plasticity in a Michigan population of Lucilia sericata (Diptera: Calliphoridae). J. Med. Entomol. 43: 1023Ð1033. Received 7 August 2009; accepted 16 June 2010.