Tropical Biomedicine 33(1): 102–108 (2016)

First record of saffranea (Diptera: ) of forensic importance in

Abd-AlGalil, F.M.A.1, Zambare, S.P.1 and Mashaly, A.M.A.2,3* 1Departments of Zoology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, India 2Department of Zoology, College of Science, King Saud University, Saudi Arabia 3Department of Zoology, Faculty of Science, Minia University, El-Minia, Egypt *Corresponding author email: [email protected] Received 12 May 2015; received in revised form 17 August 2015; accepted 18 August 2015

Abstract. Many of the blow (Calliphoridae) are of economic importance because of their effects on public health and the economy by causing in human beings and . Blow flies are also, however, forensic bioindicators since they can be used to determine the Post-Mortem Interval (PMI). Chrysomya saffranea (Bigot 1877), a dipteran calliphoridae of forensic and economic importance is currently endemic and recorded only in Australia and New Guinea. Here, the occurrence of C. saffranea is reported for the first time from Aurangabad City, Maharashtra State in India. Fully grown third instar larvae of C. saffranea were collected from decaying cats. The larvae were reared under laboratory conditions and adult flies freshly emerged from pupae were collected and identified by their morphological features.

INTRODUCTION vector of at least five helminth parasites, including Ascaris lumbricoides, and four There are about 1500 species of flies in the species of protozoan parasites: Entamoeba family Calliphoridae (Carvalho & Melo-Patiu, histolytica dispar, Entamoeba coli, Giardia 2008). The Indian representatives of this lamblia, and Cryptosporidium sp. family include 63 species under 18 genera (Getachew et al., 2007). Many Calliphorids (Nandi, 2004; Singh & Sidhu, 2004; 2007). are reported to be colonizers of human and Chrysomya is one of the most significant corpses (Carvalho & Melo-Patiu, genera of Calliphoridae with great medical, 2008). Indeed, they are one of the first veterinary and forensic importance. C. colonizer of cadavers since they are saffranea is a metallic greenish-blue blowfly attracted to the body often within minutes of with a black anterior spiracle and death (Amendt et al., 2004; Moretti et al., an appearance very similar to that of C. 2011) and lay the eggs in natural orifices or megacephala (James, 1971). wounds. The eggs develop through maggots Ten species of the genus Chrysomya and pupae from which adults then emerge. have been recorded in India (Bharti, 2011). Observation of the later stages of Some species, such as those from the genus development and the colonization sites of Chrysomya Robineau-Desvoidy, 1830, have maggots can assist in determining the manner medical relevance as vectors of pathogens, of death, the Post Mortem Interval (PMI) and including bacteria, protozoans, and any movement of the corpse from one site to helminths, and as causal agents of myiasis another after death (Moretti et al., 2011). (Greenberg, 1973). A study performed in In order to interpret a crime scene, it is Ethiopia showed that C. ruffacies acted as a essential to identify the type of species of

102 insects invading the body, their behaviours 2. Morphological description and environmental requirements (Lord & Blowfly specimens were removed from Burger, 1983). During the process of the traps daily (at 24 hour intervals), decomposition, carcasses provide a staged preserved in 70% alcohol in the field and then series of habitats for insects, each mounted on pins. The abdomens were characterized by a particular group of insects removed from both males and females and (Amendt et al., 2004; Megnin, 1894; Fuller, were then bleached unheated in 70% KOH for 1934; Payne, 1965; Sukontason et al., 2007). 24 hours, before being transferred to The process of infestation can be divided into glycerine for further dissection and three stages, entailing primary, secondary examination. Bleached abdomens and and tertiary colonizers (Fuller, 1934), genitalia were stored in micro vials with although some of the secondary colonizer glycerine and pinned beneath the respective insects have the capability to act as a primary source specimen. colonizer, such as C. rufifacies (Macquart), The procedure adopted to identify the C. saffranea (Bigot) and C. varipes morphological features and the terminology (Macquart). Studies into the ecological used in the description were based on the distribution of Calliphoridae explore whether identification key provided in (James, 1971; certain species are more associated with White et al., 1940; O’flynn & Moorhouse, 1980; particular ecological habitats (e.g. urban, Spradbery, 2002), with some modifications, forest, swamp, etc.). This article reports the which described below in the keys. The first observation of C. saffranea in India. identification characters for larvae and adults are given below. The adults and maggots were photographed using a digital MATERIALS AND METHODS Sony cyber-shot camera 16.1 MP. 5X optical zoom. 1. Collection, rearing and morphological identification 3. Key to larvae The specimens analysed in this study 1 - Tracheal trunks not heavily pigmented. were collected from the garden of the Zoology Posterior margin of segment 11 with Department at Dr Ambedkar Marathwada dorsal spines. Bands of pale brown to University Reserve (19° 522 483 N, 75° 192 dark brown spines with 1 or 2 or more 123 E), Aurangabad City, Maharashtra State teeth, occurring singly or in files. in India. The adult flies and maggots were Peritreme open. Button indistinct and in collected in July, 2012 in suspended traps open area of peritreme (Fig. 1)...... 2 placed on the body of cat died in an accident. The site was visited daily. On the second day a huge number of ants attacked the eggs and maggots and hence small amount of larvae were collected, but on the third and fourth days after its death about 40 -50 larvae which came on surface were collected. Both the adults and the maggots were reared in the laboratory in rearing boxes until the second generation. The flies were then identified by studying their morphology using a 4 stereoscope (ERMA Optical works, Tokyo, No. 44883), and a light microscope (Magnus Figure 1. C. saffranea posterior spiracle Trinocular Microscope MLX-DX, Olympus shows open peritreme with indistinct (India) PVT. LTD. No. 4B525145). button like structure

103 - Tracheal trunks not heavily pigmented, posterior margin of segment 11 without dorsal spines......

2 - Spine bands composed of elongate bifid or trifid spines anteriorly, tending to become single pointed posteriorly, whole band grading laterally into sharp thorn- like spines. Posterior sub-spiracular area bare. Anal protuberance with short files of setae along its posterior surface. Anterior spiracles with 10-11 papillae (Fig. 2)……...... Chrysomya saffranea Figure 2. Anterior spiracle of C. saffranea larvae show 11 papillae - Spine bands sparse having short blunt, simple spines mostly tending to form files of 2-5, some elongate bifid structures present in lateral regions. Posterior sub- spiracular region densely setulose. Setae forming distinct polygonal blocks which extend down posterior surface of anal protuberance. Anterior spiracles with about 13 papillae......

4. Keys to Adult 1 - Greater ampulla, in addition to the pubescence, with hairs that are longer than the height of the ampulla. Humeri not contrasting in colour with mesonotum and abdominal terga not marked with Figure 3. Anterior spiracle of adult of C. reddish yellow dorsally...... 2 saffranea, black in color

2 - Anterior spiracle black or blackish (Fig. 3). Gena orange to reddish yellow, with yellow hairs...... 3

3 - No black setulae on facial or parafacial around the vibrissa (Fig. 4), or, rarely, 2 or 3 present. Frontal stripe of female not broader at middle of frons, parallel-sided (Fig. 5). Facets of eye of male larger above than below, but without any distinct line of demarcation (Fig. 6). Squamae brown..... Chrysomya saffranea (Bigot)

- At least several, usually many, black Figure 4. Female head of C. saffranea did setulae around vibrissa (Fig. 7), on face not shows any black setulae on facial or and parafacial...... 4 parafacial around the vibrissa

104 Figure 8. Male head of C. megacephala Figure 5. Female head of C. saffranea shows demarcation between the upper with parallel frontal stripe and lower facets of eyes

Figure 6. Male head of C. saffranea without Figure 9. Female head of C. megacephala any distinct line of demarcation shows broader frontal stripe at middle of frons

4 - Squamae brown; facets of eye much enlarged above and sharply demarcated from the area of the smaller facets below (Fig. 8). Frontal stripe of female broader at middle of frons, not parallel-sided (Fig. 9) [19,20]...... Chrysomya megacephala (Fabricius)

RESULTS

The site was visited till the body get dried however rupture of the abdomen on fourth Figure 7. Female head of C. megacephala day released large number of the third instar shows many black setulae around vibrissa maggots which crawled on the surface and

105 then were collected. The bodies of the larvae DISCUSSION were smooth without prominent papillae; and few papillae were present on the posterior Calliphoridae flies are commonly known as segment. Posterior spiracles (Fig. 1) were blow flies or green bottle flies. They are small flush with the face of the posterior segment. to large metallic blue-green, robustly-built, The peritreme was open, showing the button and are covered with varying degrees of indistinctly in the open area of the peritreme, dusting. Adults frequent vegetation and especially in third instar and prepupae. excrement, or feed on decaying plant and Tracheal trunks were not heavily pigmented. animal matter. The larvae, meanwhile, are Anterior spiracles (Fig. 2) had 10-11 papillae, omnivorous, carnivorous, or parasitic. with these papillae sometimes varying from Calliphorids are known as primary one to another, and sometimes varying scavengers feeding on carrion. Although in the same larva. Spines were present on the (Wells et al., 2004) could not distinguish dorsal surface of at least a few segments, C. putoria from C. chlorogpya and C. other than the abdominal segment, and there megacephala from C. saffranea, these are were complete bands of spines on the anterior four forensically important blowflies; margins of segments to at least segment VI. therefore correct identification is a key The abdomen exhibited broad, complete criterion for PMI estimations when these flies bands of spines on II-VIII; a narrower band on are present. IX; and spines that were more lightly Nandi (2004) give the first checklist pigmented on X. On the posterior margin of record of Calliphoridae and Rhiniidae flies segment XI the band was very narrow and in India, they record about 63 species under often absent. Most spines were bifid or trifid 18 genera of Calliphoridae and 57 species interiorly. These larvae were identified as under 12 genera of Rhiniidae. This checklist larvae of C. saffranea. of Calliphoridae was updated (Bharti, 2011) Adults appeared superficially similar by giving the Rhiniiae as one of 9 subfamilies to C. bezziana and very similar to C. known in India. These subfamilies contain 30 megacephala (Fabricius). Their colour was genera and 119 species, which have been metallic blue, and there was a row of bristles recorded from all over India. at the base of the stem vein dorsally. Lower The observations reported in this study (posterior) squamae were covered with fine represent the first record of C. saffranea in hairs above, and a greater ampulla, in India, with this species having previously addition to the pubescence. Abdominal terga been reported only in Australia (Spradbery were not marked with reddish yellow dorsally 2002) and in New Guinea (James, 1971). Also while lower squamae were blackish brown called the Australian blowfly, the species has to dirty grey. The frontal stripe (Fig. 5) of infrequently been found breeding in the females was broader in the middle, parallel necrotic tissue associated with C. bezziana sided, with a relatively long ovipositor and (Villeneuve) causing myiasis, although it no marked 7 indentation in the cheek. The typically breeds on carrion. Wherever the anterior spiracle was black or blackish; gena geographic location, it has been accepted that orange to reddish with yellow hairs. Black the most significant and the first setulae (Fig. 4). on facial or parafacial around colonizers on carrion are typically species the vibrissa were absent, although rarely, two of necrophagous flies in the family’s or three black very small ones were present. Calliphoridae (blowflies) and Sarcophagidae The eye facets of males were larger above (flesh flies) (Anderson & Van Laerhoven, than below, but without any distinct line of 1996; Tabor et al., 2005). But the species demarcation. Adults were identified as of C. involved will vary according to the saffranea. biogeographic region This habit of feeding

106 on carrion means that C. saffranea is Getachew, S., Gebre-Michael, T., Erko, B., useful for PMI determination in criminal Balkew, M. & Medhin, G. (2007). Non- investigations. Many myiasis cases have biting cyclorrhaphan flies (Diptera) as been reported in India in different carriers of intestinal human parasites in metropolises caused by C. bezziana, C. slum areas of Addis Ababa, Ethiopia. megacephala and C. albiceps (Kumar et al., Acta. Tropica, 103: 186-194. 2011; Kulkarni et al., 2012; Sankari & Greenberg, B. (1973). Flies and Disease. Vol. Ramakrishnan, 2010; Radhakrishnan et al., 2. Biology and Disease Transmission. 2012; Das et al., 1979). Princeton University Press. pp. 447. James, M.T. (1971). New Species and records Acknowledgements. The authors are thankful of Australasian Calliphorinae with to Professor and Head of the department of special reference to the fauna of New Zoology of Dr. Babasaheb Ambedkar Guinea (Diptera: Calliphoridae). Pae Marathwada University for providing the Insects, 13: 1-12. laboratory facility. The authors extend their Kulkarni, S., Joshi, S., Bhalerao, A., Chopde, appreciation to the Deanship of Scientific Y. & Somalwar, S. (2012). Myiasis: A Boon Research at King Saud University for or a Bane? Case Report, Journal South providing support through the research group Asian Federation. Obstetric & Gynaeco- project no. RGPVPP- 028. logy, 4: 116-117. Kumar, V.G.S., Sowmya, G.S. & Shivananda, S. (2011). Chrysomya bezziana oral REFERENCES myiasis, Case Report, Journal Global Infectious Diseases, 3: 393-395. Amendt, J., Krette, K.R. & Zehner, R. Lord, W.D., Burger, J.F. (1983). Collection and (2004). . Natur- preservation of forensically important wiseschaften, 2: 51-65. entomological materials, Journal Anderson, G.S. & Van Laerhoven, S.L. (1996). Forensic Science, 28: 936-944. Initial studies on insect succession on Megnin, J.P. (1894). La faune des cadavres carrion in Southwestern British application de l’entomologiea la Columbia. Journal of Forensic Science, medicine legale. Encyclopedie Scien- 41: 617-625. tifique des Aide-Memoires, Masson et Bharti, M. (2011). An updated checklist of Gauthier-Villaars, Paris. 10 blowflies (Diptera: Calliphoridae) from Moretti, T.C., Bonato, V. & Godoy, W.A.C. India. Halteres, 3: 34-37 9. (2011). Determining the season of death Carvalho, C.J.B. & Melo-Patiu, C.A. (2008). from the family composition of insects Key to the adults of the most common infesting carrion, European. Journal forensic species of Diptera in South Entomology, 108: 211-218. America. Revista Brasil. Entomology. Nandi, B.C. (2004). Checklist of Calliphoridae 52: 390-406. (Diptera) of India. Record of Zoological Das, S.K., Roy, P. & Dasgupta, B. (1979). The Survey of India, Occassional, 231: 1-47. diurnal activity of O’flynn, M.A. & Moorhouse, D.E. (1980). (Macquart) (Diptera: Calliphoridae) in Identification of early immature stages Calcutta, India. Oriental Insects, 13: 149- of some common Queensland carrion 154. flies, Journal Australian Entomology Fuller, M.E. (1934). The insect inhabitants Society, 19: 53-61. of carrion: A study in animal ecology. Payne, J.A. (1965). A summer carrion study Council for Scientific and Industrial of the baby pig Susscrofa (Linnaeus). Research Bulletin, 82. Ecology, 46: 592-602.

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