Structural and Genetic Investigation of the Egg and First-Instar Larva of an Egg-Laying Population of Blaesoxipha plinthopyga (Diptera: Sarcophagidae), a Species of Forensic Importance Author(s): Meaghan L. Pimsler, Thomas Pape, J. Spencer Johnston, Robert A. Wharton, Jonathan J. Parrott, Danielle Restuccia, Michelle R. Sanford, Jeffery K. Tomberlin, and Aaron M. Tarone Source: Journal of Medical Entomology, 51(6):1283-1295. 2014. Published By: Entomological Society of America URL: http://www.bioone.org/doi/full/10.1603/ME14029 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. DIRECT INJURY,MYIASIS,FORENSICS Structural and Genetic Investigation of the Egg and First-Instar Larva of an Egg-Laying Population of Blaesoxipha plinthopyga (Diptera: Sarcophagidae), a Species of Forensic Importance MEAGHAN L. PIMSLER,1,2 THOMAS PAPE,3 J. SPENCER JOHNSTON,1 ROBERT A. WHARTON,1 JONATHAN J. PARROTT,1 DANIELLE RESTUCCIA,1 MICHELLE R. SANFORD,4 1 1 JEFFERY K. TOMBERLIN, AND AARON M. TARONE J. Med. Entomol. 51(6): 1283Ð1295 (2014); DOI: http://dx.doi.org/10.1603/ME14029 ABSTRACT Flies in the family Sarcophagidae incubate their eggs and are known to be ovoviviparous (i.e., ovolarviparous), but a laboratory-maintained colony of Blaesoxipha plinthopyga (Wiedemann) deposited clutches of viable eggs over 10 generations. A description of the egg and Þrst-instar larva of this species is provided along with genetic data (genome size and cytochrome oxidase I sequences). The egg is similar to previously described eggs of other Sarcophagidae but differs in the conÞguration of the micropyle. In the Þrst-instar larva, the oral ridges are much more developed than has been described for other species. B. plinthopyga has forensic importance, and the present descriptive information is critical for proper case management. KEY WORDS ßesh ßy, morphology, genome size, ovoviviparity While most ßies lay eggs, their strategies for optimizing position. Deposition of eggs is believed to be uncom- Þtness vary widely. Some culicine mosquitoes (Culici- mon. Oviposition has been documented in the klep- dae) deposit their eggs in rafts (Strickman and Fonseca toparasitic species Oebalia minuta (Falle´n) in the 2012), whereas other insects such as the torsalo (also subfamily Miltogramminae. In this species, females known as the human bot ßy), Dermatobia hominis (L. glue incubated eggs onto their host wasp as a means of Jr.) (Oestridae), attaches its eggs to the underside of a bringing eggs into the nest (Day and Smith 1980, porter (typically an insect such as a mosquito), which Sanborne 1982). Deposition of eggs has also been brings the eggs to the vertebrate host (Catts 1982). Flesh reported for several species in the subfamily Sar- ßies (Sarcophagidae) are well-known for using the re- cophaginae, including Sarcophaga cruentata (Mei- productive strategy of ovoviviparity (or ovolarviparity; gen), Sarcophaga exuberans (Pandelle´), Sarcophaga Ferrar 1987, Meier et al. 1999, Byrd and Castner 2010), nodosa (Engel), Sarcophaga tibialis (Macquart), and which is a reproductive strategy in which females pro- Liosarcophaga aegyptica (Salem) (Knipling 1936, Ar- duce eggs and incubate them after fertilization in a spe- thur and Coppel 1953, Aspoas 1991, Sukontason et al. cialized structure called a uterus (Abasa 1970). The eggs 2005, Salon˜ a Bordas et al. 2007, Majumder et al. 2012). hatch at deposition or are deposited in advanced em- Such eggs were not typically viable, and in these stud- bryonic development and hatch soon during or imme- ies eggs lacking well-developed embryos were prone diately following deposition (Meier et al. 1999). Vivipar- to desiccation and rarely or never hatched. Only lab- ity is a derived trait in which the eggs hatch within the oratory-bred individuals with constant access to a suit- uterus and the larvae are nourished in utero before being able breeding medium were observed to oviposit, and deposited, and is best exempliÞed in Diptera by the tsetse in all these studies oviposition was mentioned but not ßies (Glossinidae) (Tobe and Langley 1978, Cantrell investigated further; the main goals of such studies 1981, Meier et al. 1999). being the measurement or prediction of some other It is considered a ground-plan trait of Sarcophagidae life-history trait or descriptions of larval instars. that the female larviposits or ovilarviposits (Pape 1996, We describe oviposition by Blaesoxipha plinthopyga 1998). The common oviduct has a bilobed pouch (the (Wiedemann) maintained in a colony. Flies in this col- uterus) where the fertilized eggs are incubated, and ony deposited clutches of viable but not fully incubated eggs usually hatch immediately before or during de- eggs on beef liver over multiple generations. Blaesoxipha (Loew) (Sarcophagidae), in the broad sense of Pape 1 Department of Entomology, Texas A&M University, College Sta- (1994), is comprised of a large clade of koinobiont en- tion, TX. doparasitoids of mainly Orthoptera and Coleoptera 2 Corresponding author, e-mail: [email protected]. (Pape 1994) plus more basal necrophagous lineages. B. 3 Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark. plinthopyga is native to the Americas, though it was 4 Harris County Institute of Forensic Sciences, Houston, TX. recently intercepted in China (Jia et al. 2010). 0022-2585/14/1283Ð1295$04.00/0 ᭧ 2014 Entomological Society of America 1284 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 51, no. 6 Contrary to all other species of the genus, B. repeated for 10 successive generations to ensure that plinthopyga can cause myiasis and has been recorded the colony consisted of a single species. Representa- from multiple vertebrate species. In the southwestern tive samples of males, females, were collected for DNA United States, it has been reported from cases of trau- and the immature stages collected from the colony are matic injuries in spotted whiptail lizards (Cnemidophorus vouchered in the Texas A&M University Insect Col- gularis) (Whitworth and Wangberg 1985), jackrabbits lection (TAMUIC) under voucher no. 704. (Lepus californicus texianus) (Roberts 1931), and man Scanning Electron Microscopy (SEM) Sample Prep- (Patton and Evans 1929, Knipling and Rainwater 1937), aration. Samples were processed similarly to other and it has been reported as an agent of myiasis in an studies on eggs and Þrst-instar larvae (e.g., Sukontason unidentiÞed South American gecko (Marmels 1994, as et al. 2005). Freshly collected eggs were placed into a Cistudinomyia sp., corrected in Pape and Dahlem 2010). 2.5% acrolein solution to preserve and Þx them. Larval This species can occur in nuisance numbers, and can be samples were pretreated for 15Ð20 s in subboiling so common in some areas that it has been recorded that deionized water to extend the pseudocephalon before it “constantly ßies around and land on people who are Þxation and preservation in the 2.5% acrolein solution. walking in or along the edge of the desert” (Pratt 2000). Next the samples were suspended in a 1% osmium B. plinthopyga breeds in vertebrate carrion (Wells and tetroxide solution. In this study, samples were then Greenberg 1994, Vasconcelos and Araujo 2012) and has dehydrated in increasing concentrations of methanol been bred from human remains in forensic investigations (rather than ethanol) from 10 to 50% in 10% incre- (Wells and Smith 2013). ments, and then from 50 to 100% in 5% increments, A population of B. plinthopyga expressing a reproduc- followed by three Þnal washes in 100% methanol for tive trait (oviposition) was discovered on the campus of 30 min. The samples were critical-point-dried with Texas A&M University, providing a unique opportunity liquid carbon dioxide in a DCP-1 Vacuum Critical to study newly deposited eggs of a sarcophagid ßy. The Point Dryer (Denton Vacuum, Moorestown, NJ), objective of this study was to describe the egg and Þrst- mounted on 10-mm aluminum stubs with double instar larva of B. plinthopyga and provide genetic data for sticky tape, and coated by ruthenium chloride fuming. this species. Included in the genetic description is the Ruthenium chloride fuming has been shown to be a genome size of this and two related species. Previous valuable replacement for gold sputtering to increase work has demonstrated that genome size may be useful conductivity without occluding Þne detail (Dal Molin for species-level identiÞcation (Picard et al. 2012) of et al. 2011). blow ßies. Very little is currently known about the ge- Cephaloskeleton Sample Preparation. Samples of nome sizes of sarcophagids. Þrst-instar larvae were preserved as described above. Whole Þrst-instar larvae were macerated