Forensic Science International 245 (2014) 24–29 Contents lists available at ScienceDirect Forensic Science International jou rnal homepage: www.elsevier.com/locate/forsciint Effects of temperature and tissue type on Chrysomya rufifacies (Diptera: Calliphoridae) (Macquart) development a,c, b a Micah Flores *, Michael Longnecker , Jeffery K. Tomberlin a Department of Entomology, Texas A&M University, College Station, TX, USA b Department of Statistics, Texas A&M University, College Station, TX, USA c Bacterial and Rickettsial Department, Wound Infections, Walter Reed Army Institute of Research, Silver Spring, MD, USA A R T I C L E I N F O A B S T R A C T Article history: The hairy maggot blow fly, Chrysomya rufifacies (Diptera: Calliphoridae), is a forensically important fly Received 16 April 2014 often encountered on human and other vertebrate remains in temperate and tropic regions throughout Received in revised form 13 August 2014 the world including Australia, Asia, Central America and North America. C. rufifacies was reared under Accepted 18 September 2014 controlled laboratory conditions on three muscle types (i.e., porcine, equine and canine) at three Available online 13 October 2014 temperatures (i.e., 20.8, 24.8 and 28.3 8C). Rate of larval weight gain across time was statistically significant between muscle types (P 0.0001) and approaching significance across time between Keywords: temperatures (P = 0.0511). This research represents the first development study for C. rufifacies from Hairy maggot central Texas, USA and the first study to examine the impact of tissue type on its development. Blow fly Furthermore, these data, when compared to those available in the literature, indicate developmental Forensic entomology Phenotypic plasticity differences that could be due to genetic differences in populations or possibly methods employed during the studies. Caution should be emphasized when applying development data for this species from one region to forensic investigations in other ecoregions as such differences in development based on tissue fed upon by larvae, population genetics, and methodologies used in the studies could represent error in estimating the time of colonization. ß 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Type of tissue fed to immature blow flies also impacts their size and development rate [4,11]. Clark et al. [11] determined that Blow fly (Diptera: Calliphoridae) development is a quantitative Lucilia sericata (Meigen) (Diptera: Calliphoridae) larvae reared on trait; a trait that is known to vary due to genetic and environmental porcine tissue grew faster and larger than those on bovine tissue, factors [1]. Studies on blow flies have demonstrated this variation while larvae fed lung and heart of both tissue types grew faster and [2–4], but more work is needed within blow flies to understand larger than larvae fed liver. Kaneshrajah and Turner [4] recorded how single populations and multiple genotypes of the same similar results for Calliphora vicina Robineau-Desvoidy (Diptera: species respond to such conditions [5]. Calliphoridae) larvae with those fed pig lung, kidney, heart or brain Temperature is a well-recognized abiotic factor that affects growing faster and larger than those provided pig liver. Tarone and blow fly development. In most cases, warmer temperatures Foran [2] showed that, even when fed only beef liver, L. sericata accelerate development while cooler temperatures have an inverse larvae possess the potential to develop at different rates depending impact. This relationship has been documented in past growth on the experimental conditions (specifically factors affecting liver studies on blow flies at varying temperatures [6–9]. However, the moisture and the condition of the pupation substrate). A Texas amount of daily variation in temperature (i.e., temperature population of Cochliomyia macellaria (Fabricius) (Diptera: Calli- fluctuation) experienced by blow fly immatures can also influence phoridae), native to the Americas, was studied as to the effects of their rate of development, with cyclic temperatures increasing or temperature and tissue type as it relates to its development [12]. decreasing development times, depending on the species [7–10]. Temperature rather than tissue type, was determined to signifi- cantly impact Co. macellaria development (P < 0.05). Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) is an invasive blow fly species from Australia, New Zealand, New * Corresponding author at: 2475 TAMU, Department of Entomology, Texas A&M Caledonia, Samoa, Marquesas Island, Fiji, Tonga, Java, India, Ceylon University, College Station, TX 77843, USA. Tel.: +1 210 685 3931. E-mail address: [email protected] (M. Flores). (currently Sri Lanka) [13], Thailand [14], Pakistan and Iran [15]. It http://dx.doi.org/10.1016/j.forsciint.2014.09.023 0379-0738/ß 2014 Elsevier Ireland Ltd. All rights reserved. M. Flores et al. / Forensic Science International 245 (2014) 24–29 25 has been recorded in Japan, Baluchistan, Sumatra, Celebes, separated into three groups (replicates). Bovine liver was obtained Tasmania, New Hebrides, Saipan, Solomon Islands, Society from an on campus meat processing plant (E.M. ‘‘Manny’’ Islands [16], Malaysia, Calcutta [17] and was first introduced Rosenthal Meat Science and Technology Center, College Station, to Central America (Costa Rica) in 1978 where is has since moved TX, USA) and used as an oviposition medium and for rearing to Puerto Rico, Guatemala, Mexico [18], and Argentina [19]. immature flies. For each animal and pork chop package, muscle Making its way up to North America it has steadily expanded its tissue samples were placed in individual Ziploc bags, labeled and range beyond its once-believed environmental tolerance stored in a À20 8C freezer until use in the experiment. Muscle [18,20]. C. rufifacies has been documented throughout the tissue acquisition protocols were approved by the Animal Welfare contiguous United States, from California to Florida, Hawaii Assurance Program at Texas A&M University. [18] and as far north as southeastern Ontario, Canada [18,20]. C. rufifacies, like Co. macellaria, is commonly collected from animal 2.3. Development remains in central Texas [21] and is also frequently encountered on deceased humans as well (Jeffery K. Tomberlin, unpublished Methods for the development study were adapted from Byrd data). and Butler [8] and identical to those used in the Boatright and Both C. rufifacies and Co. macellaria have been hypothesized to Tomberlin [12] study. Adults (7–10 d) from the F1 generation were be ecological counterparts [22]; however each fly has evolved in provided with approximately 200 g fresh beef (bovine) liver as an different ecological conditions. As C. rufifacies is native to habitats oviposition site. Hourly observations were made for egg clutches. that experience minimal temperature variability (i.e., tropical Egg clutches less than one hour old were placed in dH2O, and region), this may have led to flies being less plastic (range of disaggregated with a camel hair brush to randomize all female egg development time responses) to variations in temperature while clutches and account for cohort variation. For each temperature 1 Co. macellaria may exhibit greater developmental plasticity as it treatment individual plastic BioQuip mosquito-breeding con- experiences a much more variable temperature range (i.e., tainer bottoms (10 [h] Â 12 cm [w]) were placed into an individual temperate region). Additionally, the tropics are renowned for sterilite plastic shoe box container (35 [l] Â 20 [w] Â 13 cm [h]) high species diversity [23], indicating that C. rufifacies may have (Townsend, MA, USA). Each plastic shoe box contained approxi- evolved under conditions that require the ability to survive on mately 500 ml (850 g) of sand (Quikrete Premium Play Sand, numerous types of carrion. If these fly species truly are ecological Atlanta, GA, USA) as pupation medium. The mosquito-breeding counterparts not only will they share the same resources and container was placed on top of the sand in the center of the environments, which have been documented previously in the shoebox. Each mosquito-breeding container held 200 g of literature [24], but they will also respond similarly to abiotic porcine, canine, or equine muscle tissue which had been cubed 3 factors. (3 cm or 25 g). Approximately 200 eggs, representing The objectives of this study were to determine the impact of multiple clutches, were placed on a moistened filter paper to temperature and muscle type on the development of a single prevent desiccation and then placed on the respective cubed population of C. rufifacies. We hypothesize that time for each stage muscle tissue in an order determined using a random number of development will be affected by the larval rearing substrate generator. Egg number was determined gravimetrically with an (muscle type) as well as the temperatures to which the flies were Adventure-Pro AV64 Ohaus scale (Pine Brook, NJ, USA). The three exposed to throughout their life cycles. replicates of each muscle tissue were placed in three stand-up environmental chambers (136LLVL Percival Scientific Inc., Perry, 2. Materials and methods IA, USA) set at 21, 24, or 27 8C with 14:10L:D and 75–80% RH using a Latin square design which assigns each replicate to one of 2.1. Fly source the three shelves without overlaps in animal tissue positions. A hobo data logger Onset (Onset Co., Pocasset, MA, USA) hobo C. rufifacies larvae (>500 individuals) were collected from U12-006 placed inside of each environmental chamber with decomposing animal remains located in College Station, TX, USA probes placed on each of the three levels of the chamber to record during July and October of 2008, May of 2009 and August of 2011 temperature hourly. and subsequent F1 generations used for necessary experiments at Eggs were monitored hourly for hatch; thereafter, observations the collection times. Larvae were brought to the Texas A&M were made every 12 h. During each observation after egg hatch, University Forensic Laboratory for Investigative Entomological three larvae of the visible cohort that approximated the largest Sciences (FLIES Facility) to initiate colonies.