Morphology of Immature Stages of Blow Fly, Lucilia Sinensis Aubertin

Acta Tropica 176 (2017) 395–401

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Acta Tropica

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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. sinensis. A: Egg showing slightly widening median area that extending almost the entire length at the midline. 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 temperature, 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

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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 tubercles (ivt). E: Surface sculpture of outer ventral tubercles (ovt) showing circular rows of small microtrichia. 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 with gold in the sputter-coating apparatus, and viewed under a 3. Results JEOL-JSM6610LV SEM (Japan) operated at 15 kV. Terminology of general larval morphology followed Courtney et al. (2000), peripheral 3.1. SEM observation of L. sinensis tubercles of larvae followed Liu and Greenberg (1989), and modiﬁca- tion of larval cephaloskeleton by Szpila et al. (2013b). 3.1.1. Egg The egg was generally creamy-white in color, elliptical in contour. 2.3. Egg measurement Freshly laid eggs are glossy. The anterior pole consists of a micropyle extremity and is somewhat slightly narrower than the posterior pole. Thirty eggs of L. sinensis were sampled and prepared for measure- Eggs were on average 1.82 ± 0.04 mm in length and 0.49 ± 0.04 mm ment. They were placed in Hoyer’s solution on a glass slide and covered across their central region (n = 30). The median area slightly widens, with a cover slip. Measurements were performed using ImageJ version expanding from the anterior pole and extending almost the entire 1.50b. length at the midline, of which 1.67 ± 0.04 mm in length and 0.03 ± 0.01 mm in width (n = 30). SEM micrograph revealed upright hatching pleats (Fig. 1A). The whole surface of the chorion is covered 2.4. Morphology of larvae under light microscopy by a reticular pattern formed by imprints of the polygonal (pentagonal or hexagonal) follicular cells, each was slightly elevated boundary Larvae of L. sinensis were sampled from the laboratory colony and (Fig. 1B). washed several times with normal saline solution. Prepared cephaloskeletons were examined and photographed under a light microscope (Olympus CX41 with Olympus DP22 digital camera). For comparison of 3.1.2. Larva the cephaloskeleton, the third instars of L. porphyrina and L. cuprina was All L. sinensis larvae were muscoid-shaped vermiform, tapered

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Fig. 3. Micrographs of third instar L. sinensis.A: Pseudocephalon showing sensory organs, and thoracic segments showing anterior spiracle (a) and spines between the 1st and 2nd thoracic segments (s). B: Light micrograph of spines between the 1st and 2nd thoracic segments. C: SEM image of spines between the 1st and 2nd thoracic segments. D: Posterior end of 8th abdominal segment displaying noticeable tubercles. ivt, inner ventral tubercles; mdt, middle dorsal tubercles; mvt, middle ventral tubercles; odt, outer dorsal tubercles; ovt, outer ventral tubercles. E: Posterior spiracles. F: Posterior spiracle showing peristigmatic tufts (pt).

anteriorly and blunt posteriorly. A pair of elongate outer ventral tu- distinguishing differences still remain, such as the antennal complex, bercles is present at the end of 8th abdominal segment (Fig. 1C). A maxillary palpus, ventral organ, oral ridges, anterior spiracle (Fig. 3A). pseudocephalon, sensory organs (antennal complex, cluster of sensilla However, extremely distinct ultrastructure of the cuticular spines be- at the maxillary palpus and ventral organ) was also apparent (Fig. 1D). tween segments is present in the third-instar. When focusing on the The first instar had few oral ridges. The anterior edge of the oral ridges dorsal spines between the 1st and 2nd thoracic segments, there are many showed numerous spines of cirri pointing downward (Fig. 1E). The rows of posteriorly-projecting acuminate spines, appear as groups, and posterior spiracle is small, consisting of two spiracles with few branches each has a pigmented single sharp tip observed under light microscope (Fig. 1F). (Fig. 3B). Similar profile of spines is seen under SEM (Fig. 3C). Tu- As for the second instar, the pseudocephalon presents similar sen- bercles at the peripheral rim of the 8th abdominal segment are greatly sory organs as the first instar, i.e., antennal complex, maxillary palpus noticeable. Similar to those the second instar, the outer ventral tu- and ventral organ (Fig. 2A). Oral ridges exhibited much differentiated bercles are markedly the longest (Fig. 3D). The posterior spiracular disc as an array of ridges between oral grooves used for channeling liquid contains three slits with almost straight slit (Fig. 3E). The ecdysial scar into the mouth opening (Fig. 2B). In the 1st thoracic segment, a pair of or button located ventromedially on the three straight spiracular slits. anterior spiracle is apparent in showing a single row of 9–12 papillae, The peristigmatic tufts adjacent to each slit are moderately branches of with 10 papillae being the most common (n = 21) (Fig. 2C). The 8th short, fine hairs (Fig. 3F). abdominal segment bears well-developed 3 pairs of tubercles – middle dorsal, outer dorsal and outer ventral tubercles; slightly developed in middle ventral tubercles, and minute in inner ventral tubercles 3.1.3. Puparium (Fig. 2D). The outer ventral tubercles are mostly elongated and pro- The puparium of L. sinensis is typical coarctate form in being cy- minent (Fig. 2D). Focusing on these tubercles, the surface of outer lindrical in shape. The anterior region is gradually rounded anteriorly ventral is covered with circular rows of small microtrichia (Fig. 2E). (Fig. 4A). Posteriorly, elongation of the outer ventral tubercles is pre- st Posteriorly, each posterior spiracular disc contains two slits (Fig. 2F). sent. On each lateral side of the 1 thoracic segment, a pair of anterior – In the third instar, the overall morphological features observed spiracles, comprising 9 12 papillae is found (Fig. 4B). At the posterior st under SEM are similar to that of the second-instar, but some important end of the 1 abdominal segment in young puparia, a clustered bubble membrane is detected dorso-laterally (Fig. 4C). Posteriorly of the 8th

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Fig. 4. Micrographs of puparium L. sinensis. A: Light micrograph showing coarctate form in having elongation of the outer ventral tubercles posteriorly (arrow). B: Anterior spiracle (a). C: Young puparia showing a clustered bubble membrane dorsolaterally of the 1st abdominal segment. D: Posterior end showing tubercles. ivt, inner ventral tubercles; mdt, middle dorsal tubercles; mvt, middle ventral tubercles; odt, outer dorsal tubercles; ovt, outer ventral tubercles. The outer ventral tubercles are mostly distinct. E: Posterior spiracles showing three straight spiracular slits and a distinct ventromedially ecdysial scar (es). F: Posterior spiracle showing spiracular slits, ecdysial scar (es) and minute peristigmatic tufts (arrows).

abdominal segment, well-developed tubercles are observed, the outer large and broad. An accessory sclerite is apparent adjacent to the base ventral tubercles being most markedly distinct (Fig. 4D). The posterior of apical mouthhooks. Dental sclerite is well-developed and separate spiracle comprises three straight spiracular slits and a distinct ventro- from the base of mouthhooks. Labial sclerite is fully developed and medially ecdysial scar (Fig. 4E). Minute peristigmatic tufts are observed apically bent downward. Intermediate sclerite is bean-shape and large. in puparium (Fig. 4F). Parastomal bar is slender and curved upward apically. Dorsal bridge is heavily sclerotized, abruptly truncated, with the same length of anterior margin of parastomal bar. The dorsal cornua, ventical plate and ventral 3.1.4. Cephaloskeleton cornua are heavily sclerotized. For comparison, the cephaloskeletons of ff The cephaloskeletons of the di erent instars of L. sinensis are shown L. porphyrina and L. cuprina was also displayed, of which the former fi in Fig. 5. In the rst instar (Fig. 5A), the cirri appear apically as nu- species showed the faint of accessory sclerite (Fig. 5D); while the latter merous backward hook, connecting to the sclerotized mouthhooks. with the absence of accessory sclerite (Fig. 5E). Labrum is faintly sclerotized. Intermediate sclerite is well-developed, connecting to the lower part of parastomal bar. The ventical plate is broad and heavily sclerotized. The dorsal cornua are slightly longer 4. Discussion than the ventral cornua. In the second instar, much differentiation of cephaloskeleton mor- Outside of molecular techniques, morphological descriptions of the phology was detected. As seen in Fig. 5B, the mouthhooks is large and immature stages of blow flies are mandatory for species identification. heavily sclerotized. Dental sclerite is developed at the bottom of As for blow flies in the genus Lucilia, although several SEM studies have mouthhooks. Parastomal bar is long, slender and slightly bent upward been performed on immature stages; egg (Liu and Greenberg, 1989; apically. The dorsal bridge lengthens apically and slightly longer than Sukontason et al., 2007b; Sanit et al., 2013), larvae (Sandeman et al., parastomal bar. Most strikingly, the dorsal cornua is much longer than 1987; Klong-klaew et al., 2012; Szpila et al., 2013a; Mendonça et al., the ventral cornua. 2014) and puparia (Sukontason et al., 2006), but no information per- The cephaloskeleton of L. sinensis is most developed in the third taining to L. sinensis was found in the literature. We herein present the instar (Fig. 5C). Not only larger in size, but it fully differentiated in first description of all immature stages of L. sinensis – egg, instars, and structure of cephaloskeleton. The posterior base of the mouthhooks is puparium.

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Table 1 Average egg size measured before staining with 1% potassium permanganate solution.

Species n Length (mm) Width (mm)

Lucilia sinensis 30 1.82 ± 0.04 0.49 ± 0.04 Lucilia cuprinaa 69 1.15 ± 0.03 0.28 ± 0.01 Lucilia porphyrinaa 31 1.45 ± 0.04 0.36 ± 0.02

a Data from Sanit et al. (2013).

Table 2 Comparison of main characteristics used to diﬀerentiate the third instar of Lucilia spp.

L. sinensisa L. porphyrinab L. cuprina

Papillae on anterior 9–12 5–94–7 spiracle Accessory sclerite at present present (faint) Absent cephaloskeleton (obvious) Tubercles at posterior present present present end (elongated (prominence) (prominence)c prominence) Peristigmatic tufts on Multi branches Numerous Multi branches posterior spiracle (+) branches (++ (++) +)

+,++,+++ Graded in number of branches. a Current study. b Data from Klong-klaew et al. (2012). c Data from Mendonça et al. (2014).

1989; Mendonça et al., 2008), L. cuprina (O’Flynn and Moorhouse, 1980; Sukontason et al., 2007b), L. porphyrina (Sanit et al., 2013). Therefore, the egg morphology is insufficient to be of value in distinguishing species separation from those related species. In Lucilia, egg size varied substantially among species. Morphometric analysis indicated eggs of L. sinensis are larger than those of L. cuprina and L. porphyrina (see Table 1). However, because the egg size among flies may depend on various factors (e.g., body mass of mother, genetic or environmental components), morphology and size of eggs may have limited usefulness for distinguishing blow flies in the genus Lucilia. The prominent elongated tubercles along the rim of the 8th abdominal segment permitted identification of L. sinensis larvae from Thailand. This character is distinct to L. sinensis. Interestingly, these tubercles begin developing in the first instar, and become much more promenent in the second and third instars, and are still noticeable in puparia. Five pairs of tubercles are apparent; two of dorsal tubercles and three of ventral tubercles, of which the outer ventral tubercles being the most markedly elongation (see Figs. 2 D, 3 D, 4 D). In L. porphyrina and L. cuprina, although a total six pairs of tubercles were observed (Fremdt et al., 2012; Klong-klaew et al., 2012; Mendonça et al., 2014), they are not elongated in L. sinensis. In this regard, such elongated tubercles were therefore allowing for differentiation among these species (Table 2). The number of papillae on the anterior spiracle is a differentiating feature among the Lucilia species in Thailand. In the current study, 9–12 Fig. 5. Light micrographs of cephaloskeleton of larvae Lucilia. A: The first instar of L. papillae observed on L. sinensis larvae differed from 6 to 9 in L. por- sinensis showing undeveloped cephaloskeleton. B: The second instar of L. sinensis showing phyrina (Klong-klaew et al., 2012) and 4–7inL. cuprina (Sukontason more differentiation. C: The third instar of L. sinensis showing fully developed. D: The et al., 2010; Mendonça et al., 2014). Interestingly, our molecular ana- third instar of L. porphyrina showing faint accessory sclerite (arrow). E: The third instar of lysis of adult blow flies using COI and COII genes in Thailand indicated L. cuprina showing absent of accessory sclerite. as, accessory sclerite; cir, cirri; db, dorsal L. sinensis is more closely related to L. porphyrina than L. cuprina (Narin bridge; dc, dorsal cornua; den, dental sclerite; is, intermediate sclerite; lb, labrum; ls, Sontigun et al., unpublished data). labial sclerite; mh, mouthhooks; pb, parastomal bar; vc, ventral cornua; vp, ventical plate. Our observations indicate the spines between the 1st and 2nd thoracic segments of L. sinensis of the third instar is greatly different from Structural features of the egg of L. sinensis found in this study are other related species in subfamily Luciliinae, such as L. cuprina, fi super cially similar to those of other Lucilia species. The slightly Hemipyrellia ligurriens (Wiedemann) (Sukontason et al., 2010). There- widening median area that extending almost the entire length at the fore, this character provides a valuable taxonomic character for species midline, upright hatching pleats and slightly elevated of the hexagonal identification (Szpila et al., 2015). pattern boundary observed in L. sinensis was similar to L. sericata Within the genus Lucilia, the illustration and micrograph of (Erzinclioglu, 1989), L. illustris, L. eximia, L. ibis (Liu and Greenberg,

400 S. Sanit et al. Acta Tropica 176 (2017) 395–401 cephaloskeletons have been described for many species, e.g., L. por- Identification of fly eggs using scanning electron microscopy for forensic investigations. Micron 39, 802–807. phyrina (Klong-klaew et al., 2012), L. cuprina (Fremdt et al., 2012), L. Mendonça, P.M., Barcosa, R.R., Carriço, C., Cortinhas, L.B., Santos-Mallet, J.R., Queiroz, illustris, L. sericata, Lucilia coeruleiviridis (Macquart) (Liu and Greenberg, M.M.C., 2014. Ultrastructure of immature stages of Lucilia cuprina (Diptera: calli- 1989), L. silvarum (Fremdt et al., 2012). The current study determined phoridae) using scanning electron microscopy. Acta Trop. 136, 123–128. Monum, T., Sukontason, K.L., Sribanditmongkol, P., Sukontason, K., Samerjai, C., the accessory sclerite is one of the characteristics that can be used to Limsopatham, K., Suwannayod, S., Klong-klaew, T., Wannasan, A., 2017. Forensically differentiate third instar Lucilia species. The accessory sclerite was ap- important blow flies Chrysomya pinguis, C. villeneuvi, and Lucilia porphyrina (Diptera: parent in L. sinensis, but is faint in L. porphyrina (Klong-klaew et al., calliphoridae) in a case of human remains in Thailand. Korean J. Parasitol. 55, 71–76. 2012), L. coeruleiviridis (Liu and Greenberg, 1989). Furthermore, the Moophayak, K., Klong-klaew, T., Sukontason, K., Kurahashi, H., Tomberlin, J.K., Sukontason, K.L., 2014. Species composition of carrion blow flies in northern accessory sclerite has not been observed in L. cuprina (see Fig. 5E; Thailand: altitude appraisal. Rev. Inst. Med. Trop. Sao Paulo 56, 179–182. Fremdt et al., 2012), L. illustris,orL. sericata (Liu and Greenberg, 1989). O'Flynn, M.A., Moorhouse, D.E., 1980. Identification of early immature stages of some fl – In conclusion, although larval morphology of Lucilia spp. is greatly common Queensland carrion ies. Aust. J. Entomol. 19, 53 61. Pohjoismäki, J.L.O., Karhunen, P.J., Goebeler, S., Saukko, P., Sääksjärvic, I.E., 2010. similar in general appearance, our results indicated that the important Indoors forensic entomology: colonization of human remains in closed environments features for differentiating L. sinensis from other Lucilia species were by specific species of sarcosaprophagous flies. Forensic Sci. Int. 199, 38–42. Sandeman, R.M., Collins, B.J., Carnegie, P.R., 1987. A scanning electron microscope prominent elongated tubercles, number of papillae in anterior spiracle, fl st nd study of L. cuprina larvae and the development of blow y strike in sheep. Int. J. spines between the 1 and 2 thoracic segments, posterior spiracle Parasitol. 17, 759–765. (peristigmatic tufts) and the morphology of the cephaloskeleton (ac- Sanit, S., Sribanditmongkol, P., Sukontason, K.L., Moophayak, K., Klong-klaew, T., cessory sclerite). The ultrastructural description of all immature stages Yasanga, T., Sukontason, K., 2013. Morphology and identification of fly eggs: application in forensic entomology. Trop. Biomed. 30, 325–337. of L. sinensis using SEM and LM in this study provides reliably useful in Shin, S.E., Lee, H.J., Park, J.H., Ko, K.S., Kim, Y.H., Kim, K.R., Park, S.H., 2015. The first the identification of flies in the genus Lucilia in Thailand and other survey of forensically important entomofauna collected from medicolegal autopsies countries where is species exists. in South Korea. BioMed. Res. Int. 2015 Article ID 606728. Skowronek, R., Tomsia, M., Szpila, K., Droździok, K., Kabiesz, J., 2015. The presence of Diptera larvae in human bones. 26th Congress of the International Society for Acknowledgements Forensic Genetics. Forensic Sci. Int. Suppl. Ser. 5, e235–e237. Sukontason, K.L., Piangjai, S., Bunchu, N., Chaiwong, T., Sripakdee, D., Boonsriwong, W., This research was supported by Chiang Mai University (CMU) Vogtsberger, R.C., Sukontason, K., 2006. Surface ultrastructure of the puparia of the blow fly, Lucilia cuprina (Diptera: Calliphoridae), and flesh fly, Liosarcophaga dux through the research administration office provided budget to our (Diptera: Sarcophagidae). Parasitol. Res. 98, 482–487. Excellence Center in Insect Vector Study, “Diamond Research Grant” of Sukontason, K., Narongchai, P., Kanchai, C., Vichairat, K., Sribanditmongkol, P., Bhoopat, the Faculty of Medicine, Chiang Mai University. This work has been T., Kurahashi, H., Chockjamsai, M., Piangjai, S., Bunchu, N., Vongvivach, S., Samai, W., Chaiwong, T., Methanitikorn, R., Ngern-klun, R., Sripakdee, D., Boonsriwong, W., presented as poster presentation at the Microscience Microscopy Siriwattanarungsee, S., Srimuangwong, C., Hanterdsith, B., Chaiwan, K., Srisuwan, Congress (MMC2017) held in Manchester, UK, on 3–6 July 2017. C., Upakut, S., Moopayak, K., Vogtsberger, R.C., Olson, J.K., Sukontason, K.L., 2007a. 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