Acta Tropica 176 (2017) 395–401 Contents lists available at ScienceDirect Acta Tropica journal homepage: www.elsevier.com/locate/actatropica Original paper Morphology of immature stages of blow fly, Lucilia sinensis Aubertin MARK (Diptera: Calliphoridae), a potential species of forensic importance Sangob Sanita, Kom Sukontasona, Hiromu Kurahashib,Jeffery K. Tomberlinc, ⁎ Anchalee Wannasana, Rungroj Kraisittipanitd, Kabkaew L. Sukontasona, a Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand b Department of Medical Entomology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan c Department of Entomology, Texas A & M University, College Station, TX, USA d Earthworm Research and Development Center, Maejo University, Chiang Mai 50290, Thailand ARTICLE INFO ABSTRACT Keywords: Lucilia sinensis Aubertin (Diptera: Calliphoridae) is a blow fly species of potential forensic importance since Lucilia sinensis adults are attracted to, and colonize, decomposing vertebrate remains. Blow fly larvae associated with human Morphology corpses can be useful evidence in forensic investigations; however, their use is dependent in most cases on proper fi Identi cation species identification and availability of developmental data. For identification, morphological information on Scanning electron microscopy each life stage is traditionally used. We used scanning electron microscopy (SEM) to examine the ultrastructure of eggs, all instars, and puparia, of L. sinensis. The important characteristics used to differentiate L. sinensis from other species are provided. Distinctive features of the eggs are the slight widening median area extending almost the entire length. The last abdominal segment of the first instar bears elongated outer ventral tubercles along the rim of the last abdominal segment. These tubercles, as well as the well developed median and outer dorsal tubercles, are more prominent in the second and third instars. The surface integument of the tubercles is equipped with circular rows of microtrichia. Pairs of inner dorsal tubercle are absent. Each anterior spiracle is comprised of 9–12 papillae arrange in a single row in the second and third instars. As for the third instar, the dorsal spines between the first and second thoracic segments are delicate, narrow, small, and close together (as row or set). The peristigmatic tufts adjacent to the posterior spiracle of the third instar are moderately branches of short, fine hairs, but minute in puparia. In conclusion, the prominent outer ventral tubercle in all instars and puparia is a new diagnostic feature of L. sinensis and helpful in differentiating it from other Lucilia species that are forensically important. The description of immature L. sinensis in this study will be useful for forensic en- tomologists in countries where this species exists. 1. Introduction et al., 2012); L. caesar Linnaeus and L. illustris (Meigen) in South Korea (Shin et al., 2015). In Thailand, a total of 27 genera and 93 species of Blow flies (Diptera: Calliphoridae) are of forensic importance since blow flies are listed, with only four Lucilia species being recorded, i.e., their larvae are often associated with human remains. As for blow flies L. cuprina, L. porphyrina, L. papuensis Macquart and L. sinensis in the genus Lucilia, L. cuprina (Wiedemann) and L. porphyrina Walker (Kurahashi and Bunchu, 2011). are forensically important in Thailand since their larvae have been In most cases, correct identification of the blow fly species is the associated with human remains (Sukontason et al., 2007a; Monum crucial first step needed for determining a time of colonization in order et al., 2017). Larvae of some of these species have been collected in to infer a minimum postmortem interval under certain assumptions human cadavers in other regions of the world; for example, L. cuprina in (e.g., colonization occurred after death). In Thailand, Lucilia is cosmo- Malaysia (Syamsa et al., 2017); L. sericata Meigen in Poland politan with known distributions in areas ranging from lowland to (Skowronek et al., 2015), Finland (Pohjoismäki et al., 2010), Italy mountainous regions (Moophayak et al., 2014). L. sinensis, which is (Bugelli et al., 2015), Japan (Toukairin et al., 2017), South Korea (Shin present in many countries of the Oriental, Australasian/Oceanian and et al., 2015); L. silvarum Meigen in Sweden, Netherland and Germany Palaearctic regions (Verves, 2005), occurs in the high forested moun- (Fremdt et al., 2012); L. ampullacea Villeneuve in Germany (Fremdt tain regions of Thailand (altitude up to 1010 m above sea level) ⁎ Corresponding author. E-mail address: [email protected] (K.L. Sukontason). http://dx.doi.org/10.1016/j.actatropica.2017.09.014 Received 25 July 2017; Accepted 16 September 2017 Available online 20 September 2017 0001-706X/ © 2017 Elsevier B.V. All rights reserved. S. Sanit et al. Acta Tropica 176 (2017) 395–401 Fig 1. SEM micrographs of egg and first instar L. si- nensis. A: Egg showing slightly widening median area that extending almost the entire length at the mid- line. B: Hexagonal pattern boundary showing slightly elevated. C: First instar showing prominent outer ventral tubercle (ovt) at the 8th abdominal segment. D: Pseudocephalon showing antennal complex (an), maxillary palpus (mp) and ventral organ (vo). E: Pseudocephalon showing ventral organ (vo), cirri (cir) and oral ridges (ori). F: Posterior spiracles (ps). (Kurahashi and Bunchu, 2011). were maintained in a transparent plastic rearing box (12 × 5 × 6 cm) To our knowledge, a morphological description of immature L. si- until they reached the third instar. Three quarters of the area of the box nensis is not available. We postulate this species is of potential forensic lid was removed and covered with a fine screen cloth for ventilation, importance since adult flies have been collected from bait traps in and to prevent entry of parasitoids. The lid was sealed tightly with Thailand (Moophayak et al., 2014); however, confirmation cannot be adhesive tape to prevent the larvae from escaping. Before pupariation, made until proper morphological descriptions are made available for sawdust soaked with water (2:1) was placed in the rearing box, which the immature stages. The objectives of this study were to, (1) describe helped to increase the relative humidity inside the container. the morphology of all immature stages (egg, larva and puparium) using Pupariation and pupation occurred inside the rearing box. Once adults scanning electron microscopy; and, (2) compare the distinctive features emerged, they were placed inside a black screened rearing cage used for differentiating from other Lucilia species. (30 × 30 × 30 cm). Adults were given sugar and water ad libitum and fresh pork liver as food and an oviposition site. Rearing and main- 2. Materials and methods tenance of the colony was performed outdoor at natural ambient tem- perature, relative humidity, and photoperiod in the year 2015. 2.1. Fly collection and rearing 2.2. Morphology of larvae under scanning electron microscopy (SEM) Specimens of L. sinensis were obtained from a laboratory colonies maintained at the Department of Parasitology, Faculty of Medicine, Larvae of all stages were sampled from the laboratory colony, and Chiang Mai University, Thailand. The F1 colony was started in 2015 killed by transferring them to a beaker containing hot water (90 °C) for from field-captured larvae collected from a decomposing earthworm, three minutes. Specimens were washed using saline solution. Each larva most probably Polypheretima elongata (Perrier) (Oligochaeta: was cut using a sharp blade at two sites; across the 2nd thoracic segment Megascolecidae) by morphological identification. Molecular methods and across the middle of the 8th abdominal segment. They were fixed were used with adult specimens of L. sinensis to confirm species (Narin with 2.5% glutaraldehyde mixed in phosphate buffer solution (PBS) at a Sontigun et al., unpublished data). pH of 7.4 at 4 °C for 24 h, rinsed twice with PBS at 10-min intervals, In the laboratory, larvae of L. sinensis were fed earthworms collected and post-fixed with 1% osmium tetroxide at room temperature for 24 h, from a forested area of the Suthep mountain (N 18°52′20.68″ E rinsed twice with PBS and dehydrated with alcohol. They were dehy- 98°54′37.64″, 683 m), Chiang Mai province, northern Thailand. Larvae drated by placing them for 12 h intervals in the following alcohol 396 S. Sanit et al. Acta Tropica 176 (2017) 395–401 Fig. 2. SEM micrographs of second instar L. sinensis. A: Pseudocephalon showing antennal complex (an), maxillary palpus (mp) and ventral organ (vo). B: Pseudocephalon showing ventral organ (vo) and oral ridges (ori). C: Anterior spiracle (a). D: Posterior end displaying middle dorsal tubercles (mdt), outer dorsal tubercles (odt), outer ventral tubercles (ovt), middle ventral tubercles (mvt) and inner ventral tu- bercles (ivt). E: Surface sculpture of outer ventral tubercles (ovt) showing circular rows of small mi- crotrichia. F: Posterior spiracle (ps). concentrations (30%, 50%, 70%, 80% and 90%). Specimens were performed in the same manner described above. Specimens for these placed in absolute alcohol for another 24 h (ethanol replaced after 12 h) two species originated from colonies maintained under similar condi- period to finalize dehydration. The larvae were subjected to critical tions as for L. sinesis in the laboratory. point drying, attached to double-stick tape on aluminum stubs and coated