Ultrastructure of male genitalia of blow flies (Diptera: Calliphoridae) of forensic importance

Acta Tropica 179 (2018) 61–80

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Original paper

Ultrastructure of male genitalia of blow flies (Diptera: Calliphoridae) of forensic importance

T

Narin Sontiguna,b, Sangob Sanita,b, Anchalee Wannasana, Kom Sukontasona, Jens Amendtc, ⁎ Tippawan Yasangad, Kabkaew L. Sukontasona, a

Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand Graduate School, Chiang Mai University, Chiang Mai 50200, Thailand c Institute of Legal Medicine, Forensic Biology/Entomology, Kennedyallee 104, 60596 Frankfurt am Main, Germany d Medical Science Research Equipment Center, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand b

A R T I C L E I N F O

A B S T R A C T

Keywords: Male genitalia Blow flies Thailand Scanning electron microscopy

Male genitalia of blow flies (Diptera: Calliphoridae) are distinctive in their morphological features and are often used for species identification. The aim of this work was to investigate the male genitalia of blow flies of medical and forensic importance from Thailand at the ultrastructural level, using scanning electron microscopy (SEM). Flies in two subfamilies were examined: Chrysomyinae [Chrysomya bezziana Villeneuve, Chrysomya chani Kurahashi, Chrysomya nigripes Aubertin, Chrysomya pinguis (Walker), Chrysomya rufifacies (Macquart), Chrysomya thanomthini Kurahashi & Tumrasvin, and Chrysomya villeneuvi Patton] and Luciliinae [Hemipyrellia ligurriens (Wiedemann), Hypopygiopsis infumata (Bigot), Hypopygiopsis tumrasvini Kurahashi, Lucilia cuprina (Wiedemann), Lucilia papuensis Macquart, Lucilia porphyrina (Walker), and Lucilia sinensis Aubertin]. Particular attention was paid to the main distinguishing features such as the shapes of the cercus and the surstylus, and the complex structure of the distiphallus. The differentiation of the male genitalia of these species at the SEM level is discussed and compared to the conditions in closely related species such as Chrysomya megacephala (Fabricius). A key for the identification of 14 blow fly species based on male genitalia is provided.

1. Introduction Blow flies are insects of medical,veterinary, and forensic importance on a global scale because of the fact that many species use breeding places that are close to human and animal dwellings. Adult blow flies are not only a nuisance as a pest but also mechanical vectors of numerous pathogens (namely viruses, bacteria, protozoan cysts, helminth eggs, and fungi) that may cause sickness in humans (Greenberg, 1971; Getachew et al., 2007; Chaiwong et al., 2012a, 2014). Their larvae can cause myiasis in living humans and animals, especially the genera Cochliomyia Townsend, Chrysomya Robineau-Desvoidy, Lucilia RobineauDesvoidy, and Calliphora Robineau-Desvoidy (Zumpt, 1965; Hall et al., 2016). Besides, immature stages of blow fly found in human corpses are important in forensic entomology because they can be used for estimating the minimum time since death (PMImin) (Lee et al., 2004; Sukontason et al., 2007; Amendt et al., 2011). In Thailand, blow flies of the subfamilies Chrysomyinae and Luciliinae have been recognized as significant insects of medical and forensic importance, including the blow fly species in the genera Chrysomya, Lucilia, Hemipyrellia

⁎

Townsend, and Hypopygiopsis Townsend (Sukontason et al., 2007; Bunchu, 2012; Chaiwong et al., 2012b). Species identification of adult blow flies based on morphology relies on the external morphological characteristics of the body and the genitalia. However, such external characteristics of the body can be highly variable, which may lead to incorrect species identification (Holloway, 1991; Williams and Villet, 2014; Grella et al., 2015). The male genitalia is one of the most important morphological characteristics for species identification due to its highly species-specific and extremely divergent form (Rognes and Paterson, 2005; Briceňo et al., 2016). Moreover, the male genitalia are often used for determining evolutionary relationships among taxa (Giroux et al., 2010; Whitmore et al., 2013; Buenaventura and Pape, 2015). Male genitalia of blow flies have been studied for species recognition under light microscopy (LM) in several species of forensic importance, for example, Chrysomya bezziana Villeneuve (Senior-White et al., 1940), Chrysomya megacephala (Fabricius) (Senior-White et al., 1940; Park, 1977), Chrysomya pinguis (Walker) (Senior-White et al., 1940; Park, 1977), Chrysomya rufifacies (Macquart) (Senior-White et al., 1940; Silva et al., 2012), Chrysomya

Corresponding author. E-mail address: [email protected] (K.L. Sukontason).

https://doi.org/10.1016/j.actatropica.2017.12.014 Received 5 September 2017; Received in revised form 4 December 2017; Accepted 11 December 2017 Available online 14 December 2017 0001-706X/ © 2017 Elsevier B.V. All rights reserved.

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2.2. Scanning electron microscopy (SEM) preparation

nigripes Aubertin (Senior-White et al., 1940), Lucilia cuprina (Wiedemann) (Senior-White et al., 1940; Park, 1977), Lucilia papuensis Macquart (Senior-White et al., 1940; Park, 1977; Tumrasvin et al., 1977), Lucilia porphyrina (Walker) (Senior-White et al., 1940; Park, 1977), Lucilia sinensis Aubertin (Senior-White et al., 1940; Tumrasvin et al., 1977), Hemipyrellia ligurriens (Wiedemann) (Senior-White et al., 1940; Park, 1977), Hemipyrellia pulchra (Wiedemann) (Senior-White et al., 1940), Hypopygiopsis infumata (Bigot) (Kurahashi, 1977), and Hypopygiopsis tumrasvini Kurahashi (Kurahashi, 1977; Moophayak et al., 2011), while a few species have been studied using scanning electron microscopy (SEM), namely C. megacephala (Chaiwong et al., 2008), Cochliomyia hominivorax (Coquerel) (Leite, 1995), and Cochliomyia macellaria (Fabricius) (Leite, 1995). Based on male genitalia, the important structures that can be used for species identification consist of the cercus, surstylus, and phallus (Senior-White et al., 1940; Kurahashi, 1977; Park, 1977). The last one, especially, is formed highly species-specific, but its structure is very complex and difficult to study under light microscopy. Consequently, SEM is the most suitable method for studying its structure. Taking into account the limited information on previous SEM studies of adult blow flies, in particular the male genitalia, this study was undertaken to illustrate the morphological structures of the male genitalia of the medically and forensically important blow fly species of Thailand. A detailed ultrastructure of this distinctive characteristic is given since it is very useful for species recognition and comparative studies between closely related species.

The abdominal segment at tergite 4th was cut from pinned specimens using a sharp scalpel under a dissecting microscope (Olympus, Japan). The obtained terminal ends were boiled in 10% potassium hydroxide (KOH) at ∼90 °C for 10 min; thereafter, they were cleaned by rinsing in 70% ethanol before dissection in a glass plate containing 70% ethanol to obtain the genitalia. For the SEM process, the specimens were completely dried at room temperature for a week. All the specimens were then attached to double-stick tape on an aluminum stub. Any debris remaining on the surface of the specimens was removed. The specimens were then coated with gold in a sputter-coating apparatus, and viewed under a JEOL-JSM6610LV scanning electron microscope (JEOL, Japan). 2.3. Terminology In this paper, the terminology of the male genitalia follows Rognes (1991), except “phallus”, which is used instead of “aedeagus”. 3. Results 3.1. General characteristics of the phallus The different characteristics of the phallus of blow flies found in Thailand between subfamilies Chrysomyinae and Luciliinae are illustrated in Fig. 1. The phallus is divided into two parts, including the short basiphallus and the elongated distiphallus. The distiphallus is the most important structure used for species identification, and it consists of the paraphallus, hypophallus, and acrophallus. The paraphallus is paired, sclerotized, and characterized by symmetrical structures, expanding from the dorsal part of the distiphallus distal to the hypophallic lobes in lateral view (Fig. 1a). It is situated between the hypophallic lobes and the acrophallus in ventral view (Fig. 1c, d) but in some species it is indistinct or even completely absent. The hypophallus is bilobed and its symmetrical structure directly expands outward from the ventral part of the distiphallus. The hypophallic lobes of Luciliinae are strongly sclerotized with a serrated ridge at the proximal part, while in Chrysomyinae it is absent (Fig. 1b, d). Therefore, the presence of the sclerotized hypophallus with its serrated ridge at the proximal part of the hypophallus lobe is the main distinguishing feature of the phallus between the blow fly species in the subfamilies Chrysomyinae and Luciliinae. The acrophallus or genital opening, allowing the sperm exit, is a semi-tubular structure, which is situated between the hypophallic lobes and is always covered with microserrations on its surface, either near to its base or near to the tip, or both (Fig. 1).

2. Materials and methods 2.1. Fly strains Males of Chrysomya chani Kurahashi, C. pinguis, Chrysomya thanomthini Kurahashi & Tumrasvin, C. (=Achoetandrus) rufifacies, Chrysomya (=Achoetandrus) villeneuvi Patton, C. (=Ceylonomyia) nigripes, L. cuprina, L. papuensis, L. porphyrina, L. sinensis, H. ligurriens, H. infumata, and H. tumrasvini used in this study were obtained through field collection during 2014–2015 and kept as pinned specimens in the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand. For C. bezziana, about 50 of the late third instar larvae were obtained from a cutaneous myiasis case in dog, investigated in the Animal hospital in Chiang Mai province. They were placed in a rearing box and sawdust was provided as a place for pupation and reared to the adult stage in the laboratory. They were identified using the taxonomic keys of . The details of the species and the collection sites are given in Table 1.

Table 1 Collection data of specimens used in this study. Subfamily

Chrysomyinae

Luciliinae

Species

Chrysomya bezziana Chrysomya chani Chrysomya nigripes Chrysomya pinguis Chrysomya rufifacies Chrysomya thanomthini Chrysomya villeneuvi Hemipyrellia ligurriens Hypopygiopsis infumata Hypopygiopsis tumrasvini Lucilia cuprina Lucilia papuensis Lucilia porphyrina Lucilia sinensis

Province (Location)

GPS reference

Chiang Mai (Muang) Lampang (Doi Khun Tan) Lampang (Doi Khun Tan) Chiang Mai (Doi Nang Kaew) Laboratory colony (origin Chiang Mai) Chiang Mai (Chom Thong, Doi Inthanon) Chiang Mai (Doi Nang Kaew) Chiang Mai (forest area, Mae Hia) Lampang (Doi Khun Tan) Chiang Mai (Sirindhorn observatory) Laboratory colony (origin Chiang Mai) Lampang (Doi Khun Tan) Chiang Mai (Doi Nang Kaew) Chiang Mai (Doi Nang Kaew)

62

Number of specimens

Latitude

Longitude

18°47′ 18°23′ 18°23′ 19°03′ 18°47′ 18°34′ 19°03′ 18°46′ 18°23′ 18°47′ 18°47′ 18°23′ 19°03′ 19°03′

98°57′ 99°12′ 99°12′ 99°22′ 98°58′ 98°28′ 99°22′ 98°56′ 99°12′ 98°55′ 98°58′ 99°12′ 99°22′ 99°22′

46“N 35“N 35“N 53“N 25“N 27“N 53“N 01“N 35“N 21“N 25“N 35“N 53“N 53“N

40“E 54“E 54“E 34“E 22“E 54“E 34“E 08“E 54“E 16“E 22“E 54“E 34“E 34“E

5 8 4 8 8 4 8 7 6 3 8 8 8 3

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Fig. 1. The SEM micrograph showing the general distinctive characteristics of the phallus of male blow flies based on the terminology of Rognes (1991). a Distiphallus of H. ligurriens, lateral view. b Distiphallus of C. pinguis, lateral view. c Distiphallus of L. sinensis, ventral view. d Distiphallus of C. thanomthini, ventral view.

3.2.1. Genus Chrysomya Robineau-Desvoidy 3.2.1.1. Chrysomya bezziana Villeneuve (Fig. 2). Viewed laterally, the cercus is broad and short, and is slightly longer than the surstylus; the surstylus is short with an oval shape (Fig. 2a). In posterior view, the cercus is short, slightly separated in the mid-line distally, and is much longer than the surstylus; the surstylus is short with an oval shape in the distal half (Fig. 2b). The distiphallus consists of numerous straight cilia along the ventral surface (Fig. 2e, f); the paraphallus is sickle-shaped, and is short and slightly curved distally (Fig. 2d, e); the hypophallic lobe is irregularly oval with a smooth external surface (Fig. 2c, d, e, f); the acrophallus has strongly sclerotized with a serrated margin on each side of the proximal part and the small tube-shaped membranous structure at the distal part, which is clearly curved toward the base of the phallus (Fig. 2e, f).

The distinctive characteristics of the male genitalia (namely, cercus, surstylus, and distiphallus) of each blow fly species are summarized in Table 2 and illustrated in Figs. 2–15.

3.2. Subfamily Chrysomyinae The shapes of the cercus and the surstylus are unique in each species, and the length of the cercus is much longer than the length of the surstylus in most species (Table 2; Figs. 2a,b to 8a,b), except for C. rufifacies which shows the length of the cercus to be slightly longer than that of the surstylus. The distiphallus shares the homology in the absence of the sclerotized with a serrated ridge at the proximal part of the hypophallic lobe. However, the morphological characteristics of the distiphallus are obviously different in different species, especially the variation in the shapes of the paraphallus, hypophallic lobe, and acrophallus.

3.2.1.2. Chrysomya chani Kurahashi (Fig. 3). Viewed laterally, the cercus and the surstylus are short with a triangular shape in the distal half and are about equal in length (Fig. 3a). In posterior view, the 63

Straight and slender

Slight constriction and bulbous distally

C. nigripes (Fig. 4)

C. pinguis (Fig. 5)

64

Straight and slender

Separated distally by two arms, sharp apically Separated distally by two arms, sharp apically Separated distally by two arms, sharp apically

H. tumrasavini (Fig. 11)

L. cuprina (Fig. 12)

L. papuensis (Fig. 13)

b

a

Terminology based on Rognes (1991). Based on Chaiwong et al. (2008).

L. sinensis (Fig. 15)

L. porphyrina (Fig. 14)

Straight and slender

H. infumata (Fig. 10)

Straight, fused distally

Slender and slightly curve distally Straight and slender

Luciliinae H. ligurriens (Fig. 9)

C. villeneuvi (Fig. 8)

C. thanomthini (Fig. 7)

Slender with a knob at the distal end Separated distally by two small rounded arms apically

Separated distally by two arms with abrupt truncation at the distal end

C. megacephalab

C. rufifacies (Fig. 6)

Triangular shape

Slightly separated in the midline distally

Cercus, shape of apical half (posterior view)

C. chani (Fig. 3)

Chrysomyinae C. bezziana (Fig. 2)

Species

Characteristic

Strongly curved inward Gradually curved inward Strongly curved inward

Straight

Slender and tapering

Straight and slender

Slender and tapering

Straight and slender

Curved sharply inward

Straight and slender

Triangular shape

Broad and tapering

Triangular shape

Triangular shape

Oval shape

Surstylus, shape of apical half (posterior view)

No

No

No

No

No

No

No

No

Yes

No

Yes

No

Yes

No

Yes

Distiphallus with numerous straight cilia along the ventral surface

Distiphallus

Sickle-shaped, long, tip expanded, with serration; strongly curved distally Sickle-shaped, long, and strongly curved distally Sickle-shaped, short, and slightly curved distally Sickle-shaped, long, and slender; strongly curved distally Sickle-shaped, long, and strongly curved distally

Sickle-shaped, long and slender, strongly curved distally No?

Spatula-shaped with serrated margin

Sickle-shaped, short, and slightly curved distally

Short and blunt apically

Sickle-shaped, short, and slightly curved distally

Indistinct

Sickle-shaped, short, and slightly curved distally

Not distinct (reduced?)

Sickle-shaped, short, and slightly curved distally

Paraphallus, shape (lateral view)

Table 2 Comparison of distinctive characteristics of male genitalia of Thai blow flies in Subfamilies Chrysomyinae and Luciliinaea.

Folded, with smooth external surface Small, flattened, with serrated margin Flattened, with serration on the surface, and curved inward Flattened, with serration on the surface Fused in unique structure with hypophallus Folded, with smooth external surface Folded, with smooth external surface

Spatula-shaped and lobed anteriorly, with smooth external surface

Rounded, with serrated margin Rounded, with smooth external surface, curved inward

Bar-shaped, with smooth external surface Rounded, with smooth external surface, curved inward

Rounded, with smooth external surface, curved inward

Rounded, with smooth external surface

Irregularly oval with a smooth external surface

Hypophallic lobe, shape (lateral and ventral views)

Short and stout, with serration on the surface; slightly curved toward the base of the phallus Straight, long, and slender, with serration on the surface Long and broad, with serration on the surface; slightly curved toward the base of the phallus Gradually broad from the base to the distal part, with serration on the surface; slightly curved toward the base of the phallus

Long and slender, with serration on the apex; moderately curved toward the base of the phallus Long and broad, with serration on the apical half, strongly curved toward the base of the phallus Short and stout with serration on the surface

Strongly sclerotized with a serrated margin on each side of the proximal part and the small and thin membranous structure at the distal part, which it is folded distally; curved toward the base of the phallus Long; strongly curved toward the distal end of the phallus

Strongly sclerotized with a serrated margin on each side of the proximal part and the small tube-shaped, membranous structure, at the distal part; curved toward the base of the phallus Strongly sclerotized with a serrated margin on each side of the proximal part and the small tube-shaped, membranous structure, at the distal part; curved toward the base of the phallus Strongly sclerotized with a serrated margin on each side of the proximal part and the small and slender, tube-shaped, membranous structure, at the distal part; curved toward the base of the phallus Moderately long; strongly curved toward the base of the phallus Strongly sclerotized with a serrated margin on each side of the proximal part and the lobe-shaped, membranous structure, at the distal part; curved toward the base of the phallus Short and straight

Acrophallus, shape (lateral and ventral views)

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3.3. Subfamily Luciliinae

cercus and the surstylus have a triangular shape, but the cercus is much longer than the surstylus (Fig. 3b). The distiphallus consists of indistinct or reduced paraphallus (Fig. 3c); rounded hypophallic lobe with a smooth external surface (Fig. 3d, e, f); the acrophallus has strongly sclerotized with a serrated margin on each side of the proximal part and the small tube-shaped membranous structure at the distal part, which is clearly curved toward the base of the phallus (Fig. 3f).

The shape of the cercus and the surstylus of blow fly species examined in this study seem to be very similar within the same genus. The distiphallus shares the homology in having the sclerotized with a serrated ridge at the proximal part of the hypophallic lobes. The distal part of hypophallic lobes show various differentiations, except for L. papuensis shows no distal projection. In genus Lucilia (L. cuprina, L. papuensis, L. porphyrina, and L. sinensis), the distiphallus has the same sickle-shaped paraphallus, but the shape and size of the hypophallic lobe and the acrophallus are different. In genus Hemipyrellia (H. ligurriens), the distiphallus is similar to the genus Lucilia, while in genus Hypopygiopsis (H. infumata and H. tumrasvini), the distiphallus is clearly different from other genera.

3.2.1.3. Chrysomya nigripes Aubertin (Fig. 4). Viewed laterally, the cercus is slender and slightly longer than the surstylus (Fig. 4a). In posterior view, the cercus is gradually tapered from the base to the distal end, and is much longer than the surstylus; the surstylus is quite slender (Fig. 4b). The distiphallus is bent in the middle and consists of an indistinct paraphallus (Fig. 4c); the bar-shaped hypophallic lobe with a smooth external surface (Fig. 4c, d); the acrophallus is moderately long and strongly curved toward the base of the phallus (Fig. 4c, d).

3.3.1. Genus Hemipyrellia Townsend 3.3.1.1. Hemipyrellia ligurriens (Wiedemann) (Fig. 9). Viewed laterally, the surstylus is long and gradually tapered to the distal end, and is slightly longer than the cercus; the cercus is slender (Fig. 9a). In posterior view, the cercus is slender and slightly curved distally, and is slightly shorter than the surstylus; the surstylus is slender and tapering from the base to the distal part (Fig. 9b). The distiphallus is similar to the L. porphyrina in having a sickle-shaped paraphallus, long and slender, which is strongly curved distally (Fig. 9c, d, e); and the hypophallic lobe has strongly sclerotized with a serrated ridge at the proximal part and folded with a smooth external surface at the distal part (Fig. 9c, e, f). The acrophallus is clearly different from L. porphyrina in that it is long and slender, with serration on the apex, and it is moderately curved toward the base of the phallus (Fig. 9c, d, e, f).

3.2.1.4. Chrysomya pinguis (Walker) (Fig. 5). Viewed laterally, the cercus is prominent, with its length more or less similar to the length of the phallus; the surstylus is short with a triangular shape (Fig. 5a). In posterior view, the cercus is gradually tapered from the base, with slight constriction in the apical half and bulbous distally, and is ∼2 times longer than the surstylus (Fig. 5b); the surstylus is short with a triangular shape (Fig. 5c). The distiphallus consists of numerous straight cilia along the ventral surface (Fig. 5e); sickle-shaped short paraphallus slightly curved distally (Fig. 5d); rounded hypophallic lobe curved inward with a smooth external surface (Fig. 5e, f); the acrophallus has strongly sclerotized with a serrated margin on each side of the proximal part and the lobe-shaped membranous structure at the distal part, which is clearly curved toward the base of the phallus (Fig. 5f).

3.3.2. Genus Hypopygiopsis Townsend 3.3.2.1 Hypopygiopsis infumata (Bigot) (Fig. 10). Viewed laterally, the cercus and the surstylus are almost equal in length (Fig. 10a). In posterior view, the cercus is straight and slender, and is slightly shorter than the surstylus; the surstylus is straight and slender (Fig. 10b). The distiphallus consists of a hypophallus lobe, which is strongly sclerotized with a serrated ridge at the proximal part and small and flattened with serration on the margin at the distal part (Fig. 10c, e, f); the paraphallus is absent. The acrophallus is long and broad, with serration on the apical half, and a large opening, and it is strongly curved toward the base of the phallus (Fig. 10c, d, f).

3.2.1.5. Chrysomya rufifacies (Macquart) (Fig. 6). Viewed laterally, the cercus and the surstylus are almost equal in length (Fig. 6a). In posterior view, the cercus is slender with a knob at the distal end, and is slightly longer than the surstylus; the surstylus is straight and slender (Fig. 6b). The distiphallus consists of short paraphallus that is blunt apically (Fig. 6c, d); rounded hypophallic lobe with serrated margin (Fig. 6c, d, f); the acrophallus is short and straight (Fig. 6d, e). 3.2.1.6. Chrysomya thanomthini Kurahashi & Tumrasvin (Fig. 7). Viewed laterally, the cercus is stout and much longer than the surstylus; the surstylus is short and stout (Fig. 7a). In posterior view, the cercus is broad in the upper half and separated distally by two arms rounded apically, and is ∼1 time longer than the surstylus; the surstylus has a broad base and is curved sharply inward in the apical half (Fig. 7b). The distiphallus consists of numerous straight cilia along the ventral surface (Fig. 7c, e); the paraphallus is sickle-shaped, and is short and slightly curved distally (Fig. 7d); the hypophallic lobe is rounded, and curved inward with a smooth external surface (Fig. 7e); the acrophallus has strongly sclerotized with a serrated margin on each side of the proximal part and the small and thin membranous structure at the distal part, which it is folded distally and it is clearly curved toward the base of the phallus (Fig. 7f).

3.3.2.2. Hypopygiopsis tumrasvini Kurahashi (Fig. 11). Viewed laterally, the cercus and the surstylus are almost equal in length; the proximal part of the surstylus is gradually curved while the distal end is sharply curved forward (Fig. 11a). In posterior view, the cercus is similar to that of H. infumata in being straight and slender in shape and slightly shorter than the surstylus, but the surstylus is slender and gradually curved inward (Fig. 11b). The distiphallus consists of a sickle-shaped paraphallus that is long, with an expanded tip with serration on the surface, and it is strongly curved distally (Fig. 11c, d, e, f); the distal hypophallic lobe is flattened with serration on the surface, and it is curved inward (Fig. 11e, f). The acrophallus is short and stout, with serration on the surface, and has a large opening (Fig. 11c, d, f). 3.3.3. Genus Lucilia Robineau-Desvoidy 3.3.3.1. Lucilia cuprina (Wiedemann) (Fig. 12). Viewed laterally, the cercus is slightly longer than the surstylus (Fig. 12a). In posterior view, the cercus is gradually tapered from the base to the distal part, and is slightly longer than the surstylus; the surstylus is straight, slightly curved inward in the middle, and quite straight in the apical half (Fig. 12b). The distiphallus consists of a sickle-shaped paraphallus, which is long and strongly curved distally (Fig. 12c, d, e); the hypophallic lobe has strongly sclerotized with a serrated ridge at the proximal part and flattened with serration on the surface at the distal part (Fig. 12c, e); the acrophallus is short and stout, with serration on

3.2.1.7. Chrysomya villeneuvi Patton (Fig. 8). Viewed laterally, the cercus is slightly longer than the surstylus (Fig. 8a). In posterior view, the cercus is straight and broad, fused distally, and much longer than the surstylus; the surstylus is straight and slender (Fig. 8b). The distiphallus consists of the spatula-shaped paraphallus with a serrated margin (Fig. 8c, e); the spatula-shaped hypophallic lobe that is lobed anteriorly with a smooth external surface (Fig. 8c, d); the acrophallus is long, with a pear-shaped opening, and it is strongly curved toward the distal end of the phallus (Fig. 8c, f). 65

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Fig. 2. The SEM micrograph of the male genitalia of C. bezziana. a Cercus and surstylus, lateral view. b Cercus and surstylus, posterior view. c Distiphallus, lateral view. d Distiphallus, dorsal view. e Distiphallus, ventral view. f Higher magnification of the acrophallus, ventral view. Abbreviations: acr, acrophallus; ce, cercus; hyl, hypophallic lobe; pp, paraphallus; su, surstylus.

the surface, and is slightly curved toward the base of the phallus (Fig. 12c, d, e, f).

straight, long, and slender with serration on the surface (Fig. 13c, d, e, f).

3.3.3.2. Lucilia papuensis Macquart (Fig. 13). Viewed laterally, the cercus is slender and slightly longer than the surstylus; the surstylus is long and gradually broadens to the distal end (Fig. 13a). In posterior view, the cercus is separated distally by two arms that are sharp apically, and is slightly longer than the surstylus; the surstylus is strongly curved inward in the apical half (Fig. 13b). The distiphallus consists of a sickle-shaped paraphallus that is short and slightly curved distally (Fig. 13c); the hypophallic lobe has strongly sclerotized with a serrated ridge and no distal projection (Fig. 13c, e); the acrophallus is

3.3.3.3. Lucilia porphyrina (Walker) (Fig. 14). Viewed laterally, the cercus is slender and slightly longer than the surstylus; the surstylus is short and broad (Fig. 14a). In posterior view, the cercus is similar to L. papuensis, but the surstylus is different in the apical half which is gradually curved inward (Fig. 14b). The distiphallus consists of a sickleshaped paraphallus, which is strongly curved distally and more slender than that of L. cuprina and L. sinensis (Fig. 14c, d); the hypophallic lobe has strongly sclerotized with a serrated ridge at the proximal part and folded with a smooth external surface at the distal part (Fig. 14c, e); the 66

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Fig. 3. The SEM micrograph of the male genitalia of C. chani. a Cercus, surstylus, and phallus, lateral view. b Cercus and surstylus, posterior view. c Distiphallus, dorsal view. d Higher magnification of acrophallus, dorsal view. e Distiphallus, ventral view. f Higher magnification of hypophallic lobe and the acrophallus, lateral view. Abbreviations: acr, acrophallus; ce, cercus; hyl, hypophallic lobe; ph, phallus; su, surstylus.

acrophallus is long and broad, with serration on the surface (Fig. 14c, d), and is slightly curved toward the base of the phallus (Fig. 14c, e, f).

surface, and is slightly curved toward the base of the phallus (Fig. 15c, e, f).

3.3.3.4. Luclilia sinensis Aubertin (Fig. 15). Viewed laterally, the cercus is slender and slightly longer than the surstylus; the surstylus is short and broad (Fig. 15a). In posterior view, the cercus and the surstylus are similar to L. papuensis (Fig. 15b). The distiphallus consists of a sickleshaped paraphallus, long and strongly curved distally (Fig. 15c, d, e); the hypophallic lobe has strongly sclerotized with a serrated ridge at the proximal part and folded with a smooth external surface at the distal part (Fig. 15c, e), which is similar to L. porphyrina; the acrophallus is gradually broad from the base to the distal part, with serration on the

3.4. Key to identify the medically and forensically important blow flies in Thailand based on male genitalia Characters used in this key are visible under a stereomicroscope as follows: 1 Hypophallic lobe without a serrated ridge proximally; acrophallus usually hidden behind the hypophallic lobes in lateral view (Fig. 1b, d) …………………………….....................................................................2 Hypophallic lobe with a serrated ridge proximally; acrophallus clearly 67

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Fig. 4. The SEM micrograph of the male genitalia of C. nigripes. a Cercus, surstylus, and phallus, lateral view. b Cercus and surstylus, posterior view. c Distiphallus, lateral view (arrows indicate indistinct paraphallus). d Distiphallus, ventral view. Abbreviations: acr, acrophallus; ce, cercus; hyl, hypophallic lobe; ph, phallus; su, surstylus.

rounded with serrated margin (Fig. 6c, d, f); acrophallus short and straight (Fig. 6c, d, e)………….........................Chrysomya rufifacies Paraphallus long, spatula-shaped with a serrated margin (Fig. 8c, e); hypophallic lobe spatula-shaped with a smooth external surface (Fig. 8c, d); acrophallus strongly curved toward the distal end of the phallus (Fig. 8c, d) …………........................................................................Chrysomya villeneuvi 8 Hypophallic lobe rounded with a smooth external surface (Fig. 3e, f); acrophallus strongly sclerotized with a serrated margin on each side of the proximal part (Fig. 3f) ………….................................Chrysomya chani Hypophallic lobe bar-shaped with a smooth external surface (Fig. 4c, d); acrophallus moderately long and strongly curved toward the base of the phallus (Fig. 4c, d) ……………………………...........Chrysomya nigripes 9 Paraphallus absent (Fig. 10c, d); hypophallic lobe small and flattened at the distal part (Fig. 10c, e, f); acrophallus long and broad, with serration on the apical half, and a large opening, strongly curved toward the base of the phallus (Fig. 10c, d, f)…...................Hypopygiopsis infumata Paraphallus sickle-shaped (Figs. 9c, 10c, 12c, 13c, 14c, 15c) …......10 10 Tip of paraphallus with serration (Fig. 11c, e); hypophallic lobe flattened with serration on the surface, curved inward (Fig. 11e); acrophallus short and stout, with serration on the surface, and a large opening (Fig. 11c, d, f).…………………….............Hypopygiopsis tumrasvini Tip of paraphallus without serration (Figs. 12 and 13c)…..................11 11 Paraphallus short (Fig. 13c); hypophallic lobe with no distal projection (Fig. 13c); acrophallus straight, long, and slender with serration on the surface (Fig. 13c, e, f)……………………...................Lucilia

seen in lateral view (Fig. 1a, c)…….......................................................9 2 Distiphallus with numerous straight cilia along the ventral surface………………….............................................................................3 Distiphalllus without numerous straight cilia along the ventral surface………………................................................................................6 3 Hypophallic lobe rounded with smooth external surface, curved inward (Figs. 5 and 7e); paraphallus sickle-shaped (Fig. 7d); acrophallus strongly sclerotized with a serrated margin on each side of the proximal part (Fig. 7f)………………………..........................................................................4 Hypophallic lobe irregularly oval with smooth external surface (Fig. 2c, d, f); paraphallus sickle-shaped (Fig. 2d); acrophallus strongly sclerotized with a serrated margin on each side of the proximal part (Fig. 2e, f) ………………..................................................................Chrysomya bezziana 4 Cercus gradually tapered from the base, with slight constriction in the apical half and bulbous distally (Fig. 5b); surstylus short with a triangular shape (Fig. 5c) ……………………………................Chrysomya pinguis Cercus enlarged at the upper half and separated distally by two arms at the lower half (Fig. 7b) …………………………........................................5 5 Terminal end of cercus abruptly truncate; surstylus stoutly triangular shaped (Chaiwong et al., 2008, see Fig. 4)...Chrysomya megacephala* Terminal end of cercus rounded; surstylus broad base, curved sharply inward in the apical half (Fig. 7b)…..........Chrysomya thanomthini 6 Paraphallus well-developed (Fig. 8c)…….…………………………..7 Paraphallus reduced or indistinct (Fig. 4c)………………………..........8 7 Paraphallus short and blunt apically (Fig. 6c, d); hypophallic lobe 68

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Fig. 5. The SEM micrograph of the male genitalia of C. pinguis. a Cercus, surstylus, and phallus, lateral view. b Cercus and surstylus, posterior view. c Surstylus, lateral view. d Distiphallus, dorsal view. e Distiphallus, ventral view. f Higher magnification of hypophallic lobe and the acrophallus, ventral view. Abbreviations: acr, acrophallus; ce, cercus; hyl, hypophallic lobe; ph, phallus; pp, paraphallus; su, surstylus.

on the surface, slightly curved toward the base of the phallus (Fig. 15c, e)………………………………………………..................................Lucilia sinensis 14 Cercus separated distally by two arms that are sharp apically (Fig. 14b); surstylus gradually curved inward (Fig. 14b) ……………………………….......................................Lucillia porphyrina Cercus slender and slightly curved distally (Fig. 9b); surstylus slender and tapering from the base to the distal part (Fig. 9b) ……………………................................................Hemipyrellia ligurriens *Male genitalia based on Chaiwong et al. (2008)

papuensis Paraphallus long (Figs. 9c, 10c, 12c, 14c, 15c); hypophallic lobe with a distal projection………………………………………………......................12 12 Distal part of hypophallic lobe flattened (Fig. 12c, e); acrophallus short (Fig. 12c, e) ………………………………………….......Lucilia cuprina Distal part of hypophallic lobe folded (Fig. 15c); acrophallus long (Fig. 9c)………...............................................................................13 13 Paraphallus great slender and strongly curved distally (Figs. 9 and 14) ……………...............................................................................14 Paraphallus broad and strongly curved distally (Fig. 15c); acrophallus gradually broad from the base to the distal part, with serration 69

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Fig. 6. The SEM micrograph of the male genitalia of C. rufifacies. a Cercus and surstylus, lateral view. b Cercus and surstylus, posterior view. c Distiphallus, lateral view. d Distiphallus, dorsal view. e Higher magnification of the acrophallus, lateral view. f Higher magnification of hypophallic lobe showing the serrated feature, and the acrophallus, ventral view. Abbreviations: acr, acrophallus; ce, cercus; hyl, hypophallic lobe; pp, paraphallus; su, surstylus.

4. Discussion

Especially regarding distiphallus morphology, the shape and size of the hypophallic lobe, paraphallus, and acrophallus can be used for species discrimination among the 14 species examined. The features of the cercus and the surstylus are often used for species identification in insects by many researchers (Senior-White et al., 1940; Park, 1977; Holloway, 1991; Leite, 1995). In this study, the shapes of the cercus and the surstylus of the Chrysomya spp. were obviously different among the species in both lateral and posterior views. The cercus within Chrysomya is much longer than the surstylus in most species, except in the case of C. rufifacies in which the cercus is slightly longer than the surstylus in posterior view and the cercus and the surstylus are almost equal in length in lateral view. Although the shapes

Identification of adult blow flies typically relies on their external morphology of the body to discriminate species; however, the genitalia is an important organ which can be used to confirm species identification when other morphological characters are indistinct or damaged. Sometimes it is the only characteristic that allows a distinction of closely related species. The present study showed the ultrastructure of the male genitalia of C. bezziana, C. chani, C. nigripes, C. pinguis, C. rufifacies, C. thanomthini, C. villeneuvi, H. ligurriens, H. infumata, and H. tumrasvini, L. cuprina, L. papuensis, L. porphyrina, and L. sinensis, highlighting in particular the shape of the cercus, the surstylus, and the distiphallus. 70

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Fig. 7. The SEM micrograph of the male genitalia of C. thanomthini. a Cercus and surstylus, lateral view. b Cercus and surstylus, posterior view. c Distiphallus, lateral view. d Distiphallus, dorsal view. e Distiphallus, ventral view. f Higher magnification of the acrophallus, ventral view. Abbreviations: acr, acrophallus; ce, cercus; hyl, hypophallic lobe; pp, paraphallus; su, surstylus.

(Rognes and Paterson, 2005), C. macellaria (Dear, 1985; Leite, 1995), C. hominivorax (Dear, 1985; Leite, 1995), Lucilia sericata (Meigen) (SeniorWhite et al., 1940; Holloway, 1991), H. pulchra (Wiedemann) (SeniorWhite et al., 1940), Hypopygiopsis fumipennis (Walker) (Kurahashi, 1977), and Hypopygiopsis violacea (Macquart) (Kurahashi, 1977). The phallus of the examined blow flies shows similar general morphology between subfamilies. The hypophallus appears to be the main distinguishing characteristic between the subfamilies Chrysomyinae and Luciliinae. The hypophallic lobes of Luciliinae are strongly sclerotized with a serrated ridge at the proximal part, while this ridge is absent in Chrysomyinae. Although the phallus morphology characteristics of blow flies in the same subfamily show similar general

of the cercus and the surstylus tend to be very similar among species within the same genus in the Luciliinae fly species examined in this study, they can be differentiated by the shape of the apical half in posterior view. In addition, the shapes of the cercus and the surstylus observed in this study are similar to those of the same species, which was previously illustrated based on LM (Senior-White et al., 1940; Kurahashi, 1977; Park, 1977; Tumrasvin et al., 1977), and shows marked differences compared to other blow fly species, for example, C. megacephala (Senior-White et al., 1940; Chaiwong et al., 2008), Chrysomya defixa (Walker) (Senior-White et al., 1940), Chrysomya phaonis (Séguy) (Senior-White et al., 1940), Chrysomya chloropyga (Wiedemann) (Rognes and Paterson, 2005), Chrysomya putoria (Wiedemann) 71

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Fig. 8. The SEM micrograph of the male genitalia of C. villeneuvi. a Cercus, surstylus, and phallus, lateral view. b Cercus and surstylus, posterior view. c Distiphallus, lateral view. d Distiphallus, dorsal view. e Higher magnification of paraphallus showing the serrated feature, dorsal view. f Higher magnification of the acrophallus, dorsal view. Abbreviations: acr, acrophallus; ce, cercus; hyl, hypophallic lobe; ph, phallus; pp, paraphallus; su, surstylus.

was found to be strongly sclerotized with a serrated margin on each side of the proximal part and the membranous structure at the distal part. All of these features revealed considerable resemblance to those observed in C. megacephala (Chaiwong et al., 2008). However, these three species could be morphologically differentiated using the feature of the distal part of the acrophallus that is distinctive in the different species, i.e., lobe-shaped in C. pinguis, small, thin and folded distally in C. thanomthini, small tube-shaped in C. bezziana, and finally small, slender and tube-shaped in C. megacephala (Chaiwong et al., 2008, Fig. 8). This feature cannot be seen using LM, but using SEM, the shape of the cercus and the surstylus clearly proves to be an significant differentiating characteristic between these four species. The features of the

appearance, they can be distinguished in closely related species and appear highly species-specific in form. In addition, the phalli in the present study was found to exhibit similar morphology with that of the same species, which was previously illustrated under LM (Senior-White et al., 1940; Kurahashi, 1977; Park, 1977; Tumrasvin et al., 1977); more details of each feature of the distiphallus are demonstrated using SEM in this study. Regarding Chrysomyinae, the phalli of C. bezziana, C. pinguis, and C. thanomthini revealed remarkably similar morphology in having numerous straight cilia along the ventral surface of the distiphallus, sickleshaped paraphallus that were short and slightly curved distally, and bilobed smooth hypophallus that curved inward; also, the acrophallus 72

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Fig. 9. The SEM micrograph of the male genitalia of H. ligurriens. a Cercus, surstylus, and phallus, lateral view. b Cercus and surstylus, posterior view. c Distiphallus, lateral view. d Distiphallus, dorsal view. e Distiphallus, ventral view. f Higher magnification of the acrophallus, ventral view. Abbreviations: acr, acrophallus; ce, cercus; hyl, hypophallic lobe; ph, phallus; pp, paraphallus; su, surstylus.

2008) observed in the current study support the close relationship between these four species, which is also consistent with their traditional classification based on adult morphology (Kurahashi, 1982) and molecular analysis (Singh et al., 2011; Zajac et al., 2016). The phalli of C. rufifacies, C. villeneuvi, and C. nigripes in this study are clearly different from those of C. bezziana, C. chani, C. pinguis, C. thanomthini, and C. megacephala (Chaiwong et al., 2008), in particular the morphological features of the hypophallic lobe, paraphallus, and acrophallus. However, they share similarities in the ultrastructure of the phallus in having no straight cilia along the ventral surface of the distiphallus and the paraphallus not being sickle-shaped. The phalli having similarities in C. rufifacies, C. villeneuvi, and C. nigripes supports

paraphallus, hypophallic lobe, and acrophallus were found to be similar to those described in the cases of the harpe, vesica, and stylus, respectively, in C. megacephala in the terminology of Senior-White et al. (1940) (Chaiwong et al., 2008). As for C. chani, although the phallus of this species shares mostly similar morphology with C. bezziana, C. pinguis, C. thanomthini, and C. megacephala (Chaiwong et al., 2008), the paraphalli are not distinct or reduced. From these overall observations, it is reasonable to assume that the phalli of C. bezziana, C. pinguis, C. thanomthini, and C. megacephala (Chaiwong et al., 2008) are closely similar to each other, but slightly different from the phallus of C. chani. In this regard, the morphological similarity of the phalli in C. bezziana, C. chani, C. pinguis, C. thanomthini, and C. megacephala (Chaiwong et al., 73

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Fig. 10. The SEM micrograph of the male genitalia of H. infumata. a Cercus, surstylus, and phallus, lateral view. b Cercus and surstylus, posterior view. c Distiphallus, lateral view. d Distiphallus, dorsal view. e Higher magnification of hypophallic lobe, ventral view. f Higher magnification of the acrophallus, ventral view. Abbreviations: acr, acrophallus; ce, cercus; hyl, hypophallic lobe; ph, phallus; su, surstylus.

between C. rufifacies and C. villeneuvi is supported by molecular studies where C. rufifacies is always placed with C. albiceps and/or C. villeneuvi (Singh et al., 2011; Zajac et al., 2016). As for C. nigripes, the degree of closeness of the genetic relationship between C. nigripes and other Chrysomya spp. remains unclear. C. nigripes was placed as a sister-group to either a clade comprising C. megacephala (Wallman et al., 2005; Nelson et al., 2007) or a clade comprising C. rufifacies (Nelson et al., 2008; Singh et al., 2011), depending on the molecular markers used. Several Chrysomya, for example, Chrysomya cabrerai Kurahashi & Salazar, Chrysomya greenbergi Wells & Kurahashi, C. phaonis, Chrysomya pacifica Kurahashi, C. chloropyga, and C. megacephala (Kurahashi and Salazar, 1977; Kurahashi, 1991; Wells and Kurahashi, 1996; Rognes

the close relationship between these three species, and reaffirms their traditional classification based on adult morphology (Kurahashi and Magpayo, 1987). The clear distinction between the phalli of C. rufifacies, C. villeneuvi, and C. nigripes makes them easily discriminable from each other. Although the phallus of C. rufifacies is obviously morphologically different from that observed in C. villeneuvi, their larvae showed the similarity of hairy appearance (Sukontason et al., 2003a, 2003b). The phallus of C. nigripes shows clear differences from the phalli of C. rufifacies and C. villeneuvi, but their larvae are smooth maggots (Sukontason et al., 2005). Based on the similarity in morphology of the larvae, it can be assumed that C. rufifacies is more closely related to C. villeneuvi than C. nigripes. The close genetic relationship 74

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Fig. 11. The SEM micrograph of the male genitalia of H. tumrasvini. a Cercus, surstylus, and phallus, lateral view. b Cercus and surstylus, posterior view. c Distiphallus, lateral view. d Distiphallus, dorsal view. e Higher magnification of paraphallus and hypophallic lobe showing the serrated feature, ventral view. f Distiphallus, ventral view. Abbreviations: acr, acrophallus; ce, cercus; hyl, hypophallic lobe; ph, phallus; pp, paraphallus; su, surstylus.

present study in which was found a sharing of similar characteristics in having numerous straight cilia along the ventral surface, as seen in C. bezziana, C. pinguis, and C. thanomthini. Therefore, this feature may be one of the characteristics for determining the phylogenetic relationship within Chrysomya spp. As for Luciliinae, the phallus of Lucilia spp. shares the general appearance, but the phalli could be differentiated in closely related species by variations in the shape and size of the paraphallus, hypophallic

and Paterson, 2005; Chaiwong et al., 2008), have distiphallus with numerous straight cilia along the ventral surface, which is similar to the current observations in C. bezziana, C. pinguis, and C. thanomthini. Based on the result from the molecular analysis of Singh et al. (2011), C. pinguis, C. thanomthini, C. megacephala, C. bezziana, C. greenbergi, C. pacifica, and C. cabrerai were placed in the same clade. This molecular analysis indicated close genetic relationship between all these species, a finding that correlates with the phallus morphology observed in the

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Fig. 12. The SEM micrograph of the male genitalia of L. cuprina. a Cercus, surstylus, and phallus, lateral view. b Cercus and surstylus, posterior view. c Distiphallus, lateral view. d Distiphallus, dorsal view. e Distiphallus, ventral view. f Higher magnification of the acrophallus, ventral view. Abbreviations: acr, acrophallus; ce, cercus; hyl, hypophallic lobe; ph, phallus; pp, paraphallus; su, surstylus.

general appearance as previously presented in other Lucilia spp. using LM, such as the hypophallus with serrated ridge and sickle-shaped paraphallus (Whitworth, 2010, 2014; Tantawi and Whitworth, 2014). The phallus of H. ligurriens is very similar to that of L. porphyrina in having a sickle-shaped paraphallus that was long and slender, the distal hypophallic lobe folded with a smooth external surface, and the acrophallus long and slender, but extremely different from the phallus of the genus Hypopygiopsis in that hypophallic lobe with serration, tip of

lobe, and acrophallus. The phalli of all the species have the same sickleshaped paraphallus, but the sizes vary. The paraphallus of L. porphyrina are strongly curved distally and more slender than those of L. cuprina and L. sinensis, whereas the paraphallus of L. papuensis is short and slightly curved distally. The features of the hypophallic lobe and the acrophallus in L. porphyrina are similar to those of L. sinensis, but L. cuprina and L. papuensis show distinctive characteristics. In the current study, the phallus of Lucilia spp. was also observed to show the same

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Fig. 13. The SEM micrograph of the male genitalia of L. papuensis. a Cercus, surstylus, and phallus, lateral view. b Cercus and surstylus, posterior view. c Distiphallus, lateral view. d Distiphallus, dorsal view. e Distiphallus, ventral view. f Higher magnification of the acrophallus, dorsal view. Abbreviations: acr, acrophallus; ce, cercus; hyl, hypophallic lobe; ph, phallus; pp, paraphallus; su, surstylus.

being slender in H. ligurriens, and the cercus is separated distally by two arms that are sharp apically and the surstylus is gradually curved inward in the apical half in L. porphyrina. The morphological similarities between the phalli of H. ligurriens and L. porphyrina observed in this study suggest that H. ligurriens is more closely related to Lucilia spp. than to Hypopygiopsis spp. This result did not correspond to the traditional classification based on adult and larval morphology in that H. tumrasvini was morphologically more similar to H. ligurriens than to L.

paraphallus with serration (H. tumrasvini) or paraphallus absent (H. infumata), and the acrophallus with distinctive serration and a large opening. However, H. ligurriens and L. porphyrina could be differentiated by the features of the acrophallus in that it is long and slender with serration on the apex in H. ligurriens and long and broad, with serration on the surface in L. porphyrina. In addition, another characteristic difference between the genitalia of these two species is in the shape of the cercus and the surstylus, which the cercus and the surstylus

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Fig. 14. The SEM micrograph of the male genitalia of L. porphyrina. a Cercus, surstylus, and phallus, lateral view. b Cercus and surstylus, posterior view. c Distiphallus, lateral view. d Distiphallus, dorsal view. e Distiphallus, ventral view. f Higher magnification of the acrophallus, ventral view. Abbreviations: acr, acrophallus; ce, cercus; hyl, hypophallic lobe; ph, phallus; pp, paraphallus; su, surstylus.

conclusion can be made regarding its relationship with Lucilia and Hemipyrellia spp. In conclusion, this study presents the ultrastructure of the external male genitalia of the medically and forensically important blow fly species of Thailand, thereby adding more morphological knowledge, which is useful for taxonomic purposes, and providing a practical key for species identification. Moreover, the findings of this study may be

cuprina (Sukontason et al., 2010; Kurahashi and Bunchu, 2011; Moophayak et al., 2011). However, based on molecular analysis, it has been demonstrated that Hemipyrellia spp. forms a clade within Lucilia spp., suggesting strong support for the synonymy of Hemipyrellia and Lucilia (McDonagh and Stevens, 2011; Williams et al., 2016). Due to the limitation sequences of Hypopygiopsis it formed a clade within both Lucilia spp. and Hemipyrellia spp. (Williams et al., 2016); thus, no

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Fig. 15. The SEM micrograph of the male genitalia of L. sinensis. a Cercus, surstylus, and phallus, lateral view. b Cercus and surstylus, posterior view. c Distiphallus, lateral view. d Distiphallus, dorsal view. e Higher magnification of hypophallic lobe, ventral view. f Higher magnification of the acrophallus, ventral view. Abbreviations: acr, acrophallus; ce, cercus; hyl, hypophallic lobe; ph, phallus; pp, paraphallus; su, surstylus.

from the Faculty of Medicine, Chiang Mai University. This work has been presented in the 8th International Congress of Dipterology, held in Potsdam, Germany, during August 10–15, 2014. We thank our anonymous reviewers for improving a first version of this manuscript.

useful for application in phylogenetic relationships: the observations on these structures may be used as a source of information on the phylogenetic characteristics and for providing the basic knowledge that may help to understand some of their functions and sexual behaviors in the future.

References Acknowledgments

Amendt, J., Richards, C.S., Campobasso, C.P., Zehner, R., Hall, M.J.R., 2011. Forensic entomology: applications and limitations. Forensic Sci. Med. Pathol. 7, 379–392. Briceňo, R.D., Eberhard, W.G., Chinea-Cano, E., Wegrzynek, D., dos Santos Rolo, T., 2016. Species-specific differences in the behavior of male tsetse fly genitalia hidden in the female during copulation. Ethol. Ecol. Evol. 28, 53–76. Buenaventura, E., Pape, T., 2015. Phylogeny of the Peckia-genus group: evolution of male genitalia in the major necrophagous of Neotropical flesh flies (Diptera:

This research was supported by the Faculty of Medicine Research Fund, Chiang Mai University, the Thailand Research Fund, through the Royal Golden Jubilee Ph.D. Program (Kabkaew L. Sukontason and Narin Sontigun; PHD/0118/2556) and the Diamond Research Grant 79

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