Acta Tropica 136 (2014) 123–128
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Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Ultrastructure of immature stages of Lucilia cuprina (Diptera: Calliphoridae) using scanning electron microscopy Paloma Martins Mendonc¸a a,b,∗ , Rodrigo Rocha Barbosa a,c , César Carric¸o a,c , Lucas Barbosa Cortinhas a,d , Jacenir Reis dos Santos-Mallet a , Margareth Maria de Carvalho Queiroz a a Laboratório de Transmissores de Leishmanioses, Setor de Entomologia Médica e Forense, Instituto Oswaldo Cruz, Fundac¸ão Oswaldo Cruz, Rio de Janeiro, Brazil b Doutoranda do Programa de Pós-graduac¸ão em Ciências Veterinárias—Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro, RJ, Brazil c Doutorando do Programa de Pós-graduac¸ão em Biologia Animal—Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro, RJ, Brazil d Mestrando do Programa de Pós-Graduac¸ão em Biodiversidade e Saúde—Instituto Oswaldo Cruz, Fundac¸ão Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
a r t i c l e
i n f o
Article history: Received 10 February 2014 Received in revised form 27 March 2014 Accepted 5 April 2014 Available online 15 April 2014 Keywords: Fly Larvae Puparia Veterinary entomology Forensic entomology SEM
a b s t r a c t The blowfly Lucilia cuprina is distributed worldwide and is a mechanical vector of pathogens. It can cause myiasis in humans and is strongly related to forensic entomology, as it is frequently found on human and animal corpses. However, most of the L. cuprina found on corpses are the immature stages of this fly. Correct identification is very important for forensic entomology but at present only the identification keys of adult L. cuprina are available. Thus, the aim of this paper was to describe and analyze the morphological characteristics of all larval instars and the puparia of L. cuprina using scanning electron microscopy (SEM).
1. Introduction Lucilia cuprina (Wiedemann, 1830) (Diptera: Calliphoridae) is a blowfly that is found worldwide and it displays typical synanthropic behavior, being intimately associated with humans and human habitations (Norris, 1965). This fly acts as a mechanical vector of pathogens, since it is associated with anthropic environments, and is frequently found on carcasses (Ferreira and Lacerda, 1993; Linhares, 1981). It is an insect of great medical and veterinary importance as it can cause myiasis in humans and in animals, principally in sheep. This fly is commonly known as the Australian sheep blowfly and can have an economic impact on the sheep industry (Stevens and Wall, 1996; Zumpt, 1965).
∗ Corresponding author at: Laboratório de Transmissores de Leishmanioses, Setor de Entomologia Médica e Forense, Instituto Oswaldo Cruz, Fundac¸ão Oswaldo Cruz, Av. Brasil, 4365—Manguinhos, Rio de Janeiro, RJ, Brazil. Tel.: +55 21 2562 1846/+55 2198495591. E-mail address: palomamm@ioc.fiocruz.br (P.M. Mendonc¸a). http://dx.doi.org/10.1016/j.actatropica.2014.04.007 0001-706X/© 2014 Elsevier B.V. All rights reserved.
© 2014 Elsevier B.V. All rights reserved.
Lucilia cuprina is considered an important forensic specie, since it has been frequently found associated with human and animal corpses according to many authors (Byrd and Castner, 2001; Early and Goff, 1986; Goff, 2000; Greenberg and Kunich, 2002; Smith, 1986). This fly is the dominant specie during the active decomposition stage, and is responsible for the removal of most of the tissue (Early and Goff, 1986). The blowflies (Diptera: Calliphoridae) are the first insects to arrive at a dead body and they readily oviposit on it (Smith, 1986). Therefore, knowledge of the length and activity of the larval stages of these flies can help determine the postmortem interval (Erzinclioglu, 1989). Most of these insects found on carcasses are the immature forms, and in order to help entomologists estimate the postmortem interval the correct identification of the immature forms is very important. The immature forms collected from corpses are commonly preserved in ethanol for later identification. However, there is a need to rear these larvae until adults in order to identify and analyze all the immature stages. The calliphorid larvae are very similar and some authors have described the first and third instar of L. cuprina using light microscopy focusing on the cephalopharyngeal skeleton, which is
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Fig. 1. Scanning electron micrographs of a first instar larva of Lucilia cuprina (Diptera: Calliphoridae). (A) Larval body with groups of spines located between the segments (arrows); the anterior (ae) and posterior ends (pe) of the larval body (×110). (B) Cephalic region with antennae (a), maxillary palps (mp) and bucal hooks (arrow) (x900). (C) Detail of spines between the first segment and thoracic region (×1100). (D) Anal segment with anal tubercles and posterior spiracles (arrows) (×500). (E) Posterior spiracles (arrow) (×1900).
the main structure visible using this technique (Sukontason et al., 2010; Szpila et al., 2013). However, light microscopy does not provide details of other characteristics that could have a diagnostic value (Liu and Greenberg, 1989; Mendonc¸a et al., 2010). Thus, the aim of this study was to describe and analyze the morphological characteristics of all larval instars and puparia of L. cuprina using scanning electron microscopy (SEM) in order to elucidate new diagnostic features of this specie.
and examined under a Jeol JSM 6390LV scanning electron microscope (Akishima, Tokyo, Japan). The SEM images were transferred directly to a computer. Puparia were not submitted to any kind of chemical fixation. Puparia were put under refrigeration for 5 min and then they were placed onto double-stick tape on metallic supports, coated with gold and examined under the same SEM. 3. Results
2. Material and methods 3.1. First larval instar The immature samples of L. cuprina used in this study were collected in Macapá, Amapá State, Brazil, using pig carcasses as bait, according to Barbosa et al. (2009). The colonies were reared and maintained at the Setor de Entomologia Médica e Forense, which is part of the Laboratório de Transmissores de Leishmanioses, Instituto Oswaldo Cruz, Fundac¸ão Oswaldo Cruz, in the state of Rio de Janeiro, as previously described (Queiroz and Milward-de-Azevedo, 1991). Insects were kept in cages at room temperature and supplied ad libitum with water and sugar. Bovine meat was used as a source of protein to stimulate oviposition and as food for the immature larvae. The third generation of the laboratory colony was used in this study. Ten specimens of each immature stage were analyzed. The terminology used in describing the morphology in this paper followed Margaritis (1985) and Mc Alpine et al. (1981). The three larval instar forms were washed several times in distilled water. Larvae were killed by placing them in hot water for 5 min and then they were fixed in 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer, pH 7.2, for 1 h and postfixed in 1% osmium tetroxide in the same buffer for 1 h at room temperature in the dark. Then these immature forms were washed with sodium cacodylate buffer three times for 10 min each. Afterward, they were dehydrated in an ascending series of acetone up to 100% and submitted to the critical point drying method, using superdry CO2 in a Balzers apparatus (Hayat, 1970). Specimens were placed on metallic supports, coated with a thin layer of gold (20–30 nm)
The larval body of L. cuprina is composed of 12 segments (one cephalic; three thoracic and eight abdominal) and has the vermiform shape typically found in muscoids (Fig. 1A). The anterior end, comprising the cephalic region, is pointed and the posterior end is blunt. The larval body is 3.76 ± 0.27 mm long and 0.199 ± 0.037 mm wide. The cephalic region is slightly bilobed and bears the sensorial structures: a pair of antennae and a pair of maxillary palps. Because of its localization, antennae are also known as dorsal organs and maxillary palps are known as terminal organs. The maxillary palps are formed by a complex of sensitive papillae. This cephalic region also includes the buccal hooks and oral cristae, but these structures are not well developed in the first instar (Fig. 1B). There is a band of spines between the cephalic region and the first thoracic segment. Two types of spines were observed: one type is flattened with tapered ends and the other is narrower with tapered tips, all of them are projected backward (Fig. 1C). The body tegument is smooth with the intersegmental line similar to those of the first thoracic segment. In this larval instar, the anterior spiracle is not visible. The posterior end or anal segment is covered with filiform spines and only the anal tubercles can be seen easily; the other tubercles are too small to be seen. The posterior spiracle is located at the top of an elevation and the single spiracular or peritrema opening is sustained by spiracular muscles (Fig. 1D and E).
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Fig. 2. Scanning electron micrographs of a second instar larva of Lucilia cuprina (Diptera: Calliphoridae). (A) Larval body with groups of spines located between the segments (arrows); the anterior (ae) and posterior ends (pe) of the larval body (×43). (B) Cephalic region with antennae (a), maxillary palp (mp), spines between the first thoracic segment and cephalic region (cs), oral cristae (oc), bucal hooks (bh) and anterior spiracles (as) (×180). (C) Details of antennae (a) and maxillary palps with papillae (mp) (×950). (D) Antennae (a), maxillary palps (mp) and spines between the segments (cs) (×700). (E) Details of anterior spiracles in a row (×850). (F) Posterior spiracles at posterior end (ps) and anal tubercles (at) (×170).
3.2. Second larval instar The body shape of the second larval instar is very similar to the first larval instar. The anterior end is pointed and the posterior end is blunt (Fig. 2A). The normal sizes of second instar larvae are 4.35 ± 0.27 mm in length and 0.624 ± 0.085 mm in width. The body tegument is smooth and the intersegmental spines are similar to those from the first thoracic segment and more visible than in the first instar. At the cephalic region, the bilobed shape is more pronounced and the antennae, maxillary palps and buccal hook are more developed than in the first instar (Fig. 2B and C). The band of spines from the first thoracic segment is flattened as found in the first instar, but the tips are not slender (Fig. 2D). The narrower spines were not observed in the second instar. The anterior spiracle, located in the first thoracic segment, is visible and well developed; it is composed of a row of 6–7 spiracular ramifications (Fig. 2E). The posterior end is still covered with filiform spines and the anal tubercles are more visible (Fig. 2F). A pair of posterior spiracles with two spiracular openings could be seen sustained by spiracular muscles (Fig. 2F). The peritreme is complete with an ecdysial scar (Fig. 2F).
posterior spiracle (Fig. 3F). Each posterior spiracle has three spiracular openings which are sustained by the spiracular muscles (Fig. 3G). The peritreme is still complete with an ecdysial scar (Fig. 3G). 3.4. Puparia In the puparia the anterior end is retracted and the posterior end is blunted (Fig. 4A). The puparia are 7.74 ± 0.67 mm in length and 2.18 ± 0.62 mm in width. The bands of spines limiting the segments are clearly visible. The spines are larger and flattened with slender tips like the third instar larvae. In the puparia of L. cuprina, there is a longitudinal ridge, which is a continuous line along the second and third thoracic segments (Fig. 4A). The anterior spiracle is located at the most anterior point of the cephalic region and is composed of 6–7 spiracular ramifications (Fig. 4B and C). At the end of the first abdominal, a tubular respiratory horn with longitudinal respiratory openings is visible (Fig. 4D). Each posterior spiracle, like the third instar larvae, has three spiracular openings with a clearly visible radial form and an opened peritreme without a scar (Fig. 4E). 4. Discussion
3.3. Third larval instar The third larval instar is very similar to the other two instars but all cephalic structures are fully developed (Fig. 3A and B). This larvae is 9.18 ± 0.19 mm long and 1.432 ± 0.7 mm wide. The band of spines from the first thoracic segment are flattened and the tips are very small (Fig. 3C). The intersegmental spines are large and flattened with slender tips and there are also some smaller spines with almost blunt tips (Fig. 3D). The anterior spiracle is composed of a row of 6–7 spiracular ramifications (Fig. 3E). The filiform spines in the anal segment are smaller than in the other two instars. The anal, ventral and dorsal tubercles are more visible and are important to protect the
The larval bodies of all muscoids are very similar. The vermiform shape found in L. cuprina is typical. This fly shares some characteristics with others blowflies of the same genus. In Latin America, the genus Lucilia blowfly is always collected rearing on carcasses, and in colder weather, it is one of the first to oviposit and rear on a dead body (Barreto et al., 2002; Carvalho et al., 2000; Ortloff et al., 2012; Salviano et al., 1996). Some authors have related the difficulties to identify the larvae using light microscopy (Oliveira et al., 2007; Queiroz et al., 1997), and the problems to rear these flies until adults in order to be able to use the taxonomic keys available. So, the use of scanning electron microscopy could provide diagnostic characteristics to help
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Fig. 3. Scanning electron micrographs of a third instar larva of Lucilia cuprina (Diptera: Calliphoridae). (A) Larval body with groups of spines located between the segments (arrows); the anterior (ae) and posterior ends (pe) of the larval body (×14). (B) Cephalic region with spines between the first segment and thoracic region (cs), antennae (a), maxillary palp (mp), oral cristae (oc) and bucal hooks (bh) (×170). (C) Detail of spines between the first segment and thoracic region (×400). (D) Detail of spines between the body segments (×280). (E) Details of anterior spiracles in a row (×550). (F) Anal segment with posterior spiracle (ps) dorsal tubercles (dt), ventral tubercles (vt) and anal tubercles (at) (×43). (G) Posterior spiracles with three spiracular openings (arrows) and ecdysial scar (s) (×160).
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Fig. 4. Scanning electron micrographs of a puparia of Lucilia cuprina (Diptera: Calliphoridae). (A) Puparia composed of groups of spines located between the segments (arrows); the anterior (ae) and posterior ends (pe) of the puparia; longitudinal ridge (dotted arrow); respiratory horn (circle) (×30). (B) Anterior region retracted (arrow) and anterior spiracle (ae) (×75). (C) Details of anterior spiracles in a row (×450). (D) Detail of spines between the body segments (s); respiratory horn (rh) (×450). (E) Posterior spiracles with three spiracular openings (arrows) and without an ecdysial scar (s) (×120).
entomologist identify the flies (Liu and Greenberg, 1989; Mendonc¸a et al., 2008, 2012a,b, 2013). Sandeman et al. (1987) described some characteristics of the larval instar of L. cuprina used from experimental infections with sheep. These authors focused on the mouth parts to verify if the first larval instar is able to damage sheep skin. As observed by these authors, the buccal hooks of the first instar larvae are very short but these parts are more developed on the second and third instar and are essential for feeding and locomotion. Apparently, there is no structural alteration among larvae reared on artificial diets and natural lesions. More recently, Szpila et al. (2013) analyzed six species belonging to the genus Lucilia in Europe, but they focused their work on the first instar larvae. The general morphology of the larvae is very similar to our observations. These authors used the spinulation of the abdominal segment A7 to distinguish between L. cuprina and L. sericata, as this is the main taxonomic question in Europe. Here, in Brazil, L. sericata is restricted to the south of the country and the
adult taxonomy is well established. However, due to the absence of studies comparing the larval morphology of the three species of the genus in Brazil, the doubts concerning the spinulation remain. In the L. cuprina collected in Amapá State, in the north of Brazil, the spinulation of segment A7 is incomplete, as related by Szpila et al. (2013). Although, the possibility of other arrangements could exist, since Wallman et al. (2005a,b) related the probable existence of cryptic species in L. cuprina. The number of spiracular ramifications is one of the most important characters to distinguish among dipterans species (Guimarães and Amorim, 2006). Sukontason et al. (2010) observed 4–7 openings at L. cuprina from Thailand using light microscopy. Sandeman et al. (1987) did not mention these structures. In the Brazilian L. cuprina species, the number of spiracular openings observed was 6 or 7. Greenberg and Singh (1995) related some morphological characteristics that could vary among dipteran species. These variations could reflect the alterations that have occurred to the species along
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the years in order to adapt to new habitats. In this work scanning electron microscopy has shown several characteristics that can be used as baseline data to help entomologists identify the immature forms in forensic investigations present in each habitat. Acknowledgments We would like to thank the Platform Electron Microscopy Rudolf Barth of Instituto Oswaldo Cruz (FIOCRUZ) for the use of the scanning electron microscope. This work was supported by grants from Instituto Oswaldo Cruz (IOC/FIOCRUZ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (Capes). References Barbosa, R.R., Mello-Patiu, C.A., Mello, R.P., Queiroz, M.M.C., 2009. New records of calyptrate dipterans (Fanniidae, Muscidae and Sarcophagidae) associated with the decomposition of domestic pigs in Brazil. Mem. Inst. Oswaldo Cruz 104, 923–926. Barreto, M., Burbano, M.E., Barreto, P., 2002. Flies (Calliphoridae, Muscidae) and beetles (Silphidae) from human cadavers in Cali, Colombia. Mem. Inst. Oswaldo Cruz 97 (1), 137–138. Byrd, J.H., Castner, J.L., 2001. Forensic Entomology: The Utility of Arthropods in Legal Investigations. CRC Press, Boca Raton, FL. Carvalho, L.M.L., Thysen, P.J., Linhares, A.X., Palhares, F.A.B., 2000. A checklist of arthropods associated with pig carrion and human corpses in southeastern Brazil. Mem. Inst. Oswaldo Cruz 95 (1), 135–138. Early, M., Goff, M.L., 1986. Arthropod succession patters in exposed carrion on the Island of O’Ahw, Hawaiian Island, USA. J. Med. Entomol. 23, 520–531. Erzinclioglu, Y.Z., 1989. The value of chorionic structure and size in the diagnosis of blowfly eggs. Med. Vet. Entomol. 3, 281–285. Ferreira, M.J.M., Lacerda, P.V., 1993. Muscoides sinantrópicos associados ao lixo urbano de Goiânia—GO. Rev. Bras. Zool. 10 (2), 185–195. Goff, M.L., 2000. A Fly for the Prosecution: How Insect Evidence Helps Solve Crimes. Harvard University Press, London, UK. Greenberg, B., Kunich, J.C., 2002. Entomology and the Law: Flies as Forensic Indicators. Cambridge University Press, England. Greenberg, B., Singh, D., 1995. Species identification of calliphorid (Diptera) eggs. J. Med. Entomol. 32, 21–26. Guimarães, J.H., Amorim, D.S., 2006. Diptera. In: Costa, C., Ide, S., Simonka, C.E. (Eds.), Insetos Imaturos: Metamorfose e Identificac¸ão. Holos, São Paulo, pp. 147–160. Hayat, M.A., 1970. Principles and Techniques of Electron Microscopy. Biological Applications. Van Nostrand Reinhold Company, New York, NY. Linhares, A.X., 1981. Sinanthropy of calliphoridae and sarcophagidae (Diptera) in the city of Campinas, São Paulo, Brazil. Rev. Bras. Entomol. 25 (3), 189–215. Liu, D., Greenberg, B., 1989. Immature stages of some flies of forensic importance. Ann. Entomol. Soc. Am. 82, 80–93. Margaritis, L.H., 1985. Structure and physiology of eggshell. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology. v.I. Embryogenesis and Reproduction. Pergamon Press, Oxford, UK, pp. 151–230.
Mc Alpine, J.F., Peterson, B.V., Shewell, G.E., Teskey, J.H., Vockeroth, J.R., Wood, D.N., 1981. Manual of Neartic Diptera. Research Branch Agriculture Canada, Ottawa. Mendonc¸a, P.M., Santos-Mallet, J.R., Mello, R.P., Gomes, L., Queiroz, M.M.C., 2008. Identification of fly eggs using scanning electron microscopy for forensic investigations. Micron 39, 802–807. Mendonc¸a, P.M., Santos-Mallet, J.R., Queiroz, M.M.C., 2010. Ultramorphological characteristics of immature stages of Chrysomya albiceps (Wiedemann, 1819) (Diptera: Calliphoridae), a fly specie of forensic importance. Microsc. Res. Tech. 73, 779–784. Mendonc¸a, P.M., Santos-Mallet, J.R., Queiroz, M.M.C., 2012a. Ultrastructure of larvae and puparia of the blowfly Chrysomya megacephala (Diptera: Calliphoridae). Microsc. Res. Tech. 75, 935–939. Mendonc¸a, P.M., Santos-Mallet, J.R., Queiroz, M.M.C., 2012b. Ultrastructure of immature stages of the blowfly Chrysomya putoria (Wiedemann, 1818) (Diptera: Calliphoridae). Microsc. Res. Tech. 75, 206–211. Mendonc¸a, P.M., Barbosa, R.R., Cortinhas, L.B., Santos-Mallet, J.R., Queiroz, M.M.C., 2013. Ultrastructure of immature stages of Peckia (Euboetcheria) collusor (Diptera: Sarcophagidae). Acta Trop. 128 (3), 522–527. Norris, K.R., 1965. The bionomics of blowflies. Ann. Rev. Entomol. 10, 47–68. Oliveira, M.S., Mello, R.P., Queiroz, M.M.C., 2007. Morfologia e durac¸ão dos instares larvais de Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae), em laboratório. Rev. Bras. Biol. 51, 239–245. ˜ P., Riquelme, M., 2012. Preliminary study of the succession pattern Ortloff, A., Pena, of necrobiont insects, colonising species and larvae on pig carcasses in Temuco (Chile) for forensic applications. Forensic Sci. Int. 222 (1-3), e36–e41. Queiroz, M.M.C., Milward-de-Azevedo, E.M.V., 1991. Técnicas de criac¸ão e alguns aspectos da biologia de Chrysomya albiceps (Wiedemann) (Diptera, Calliphoridae), em condic¸ões de laboratório. Rev. Bras. Zool. 8, 75–84. Queiroz, M.M.C., Mello, R.P., Lima, M.M., 1997. Morphological aspects of the larval instars of Chrysomya albiceps (Diptera: Calliphoridae) reared in the laboratory. Mem. Inst. Oswaldo Cruz 92, 187–196. Wallman, J.F., Leys, R., Hogendoorn, K., 2005a. Molecular systematics of Australian carrion-breeding blowflies (Diptera: Calliphoridae) based on mitochondrial DNA. Invertebr. Syst. 19, 1–15. Salviano, R.J.B., Mello, R.P., Beck, L.C.N.H., Ferreira, A., 1996. Calliphoridae (Diptera) associated with human corpses in Rio de Janeiro, RJ, Brazil. Entomol. y Vect. 3, 145–146. Sandeman, R.M., Collins, B.J., Carnegie, P.R., 1987. A scanning electron microscope study of Lucilia cuprina larvae and the development of blowfly strike in sheep. Int. J. Parasitol. 17 (3), 759–765. Smith, K.G.V., 1986. A Manual of Forensic Entomology, British Museum (Natural History). Cornell University, London. Stevens, J., Wall, R., 1996. Classification of the genus Lucilia (Diptera: Calliphoridae): a preliminary parsimony analysis. J. Nat. Hist. 30, 1087–1094. Sukontason, K., Sribanditmongkol, P., Ngoen-klan, R., Klong-klaew, T., Moophayak, K., Sukontason, K.L., 2010. Differentiation between Lucilia cuprina and Hemipyrellia ligurriens (Diptera: Calliphoridae) larvae for use in forensic entomology applications. Parasitol. Res. 106, 641–646. Szpila, K., Hall, M.J.R., Pape, T., Grzywacz, A., 2013. Morphology and identification of first instars of the European and Mediterranean blowflies of forensic importance. Part II. Luciliinae. Med. Vet. Entomol. 27 (4), 349–366. Zumpt, F., 1965. Myiasis in Man and Animals in the Old World. Butterworths, London. Wallman, J.F., Leys, R., Hogendoorn, K., 2005b. Molecular systematics of Australian carrion-breeding blowflies (Diptera: Calliphoridae) based on mitochondrial DNA. Invertebr. Syst. 19, 1–5.
Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Ultrastructure of immature stages of Lucilia cuprina (Diptera: Calliphoridae) using scanning electron microscopy Paloma Martins Mendonc¸a a,b,∗ , Rodrigo Rocha Barbosa a,c , César Carric¸o a,c , Lucas Barbosa Cortinhas a,d , Jacenir Reis dos Santos-Mallet a , Margareth Maria de Carvalho Queiroz a a Laboratório de Transmissores de Leishmanioses, Setor de Entomologia Médica e Forense, Instituto Oswaldo Cruz, Fundac¸ão Oswaldo Cruz, Rio de Janeiro, Brazil b Doutoranda do Programa de Pós-graduac¸ão em Ciências Veterinárias—Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro, RJ, Brazil c Doutorando do Programa de Pós-graduac¸ão em Biologia Animal—Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro, RJ, Brazil d Mestrando do Programa de Pós-Graduac¸ão em Biodiversidade e Saúde—Instituto Oswaldo Cruz, Fundac¸ão Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
a r t i c l e
i n f o
Article history: Received 10 February 2014 Received in revised form 27 March 2014 Accepted 5 April 2014 Available online 15 April 2014 Keywords: Fly Larvae Puparia Veterinary entomology Forensic entomology SEM
a b s t r a c t The blowfly Lucilia cuprina is distributed worldwide and is a mechanical vector of pathogens. It can cause myiasis in humans and is strongly related to forensic entomology, as it is frequently found on human and animal corpses. However, most of the L. cuprina found on corpses are the immature stages of this fly. Correct identification is very important for forensic entomology but at present only the identification keys of adult L. cuprina are available. Thus, the aim of this paper was to describe and analyze the morphological characteristics of all larval instars and the puparia of L. cuprina using scanning electron microscopy (SEM).
1. Introduction Lucilia cuprina (Wiedemann, 1830) (Diptera: Calliphoridae) is a blowfly that is found worldwide and it displays typical synanthropic behavior, being intimately associated with humans and human habitations (Norris, 1965). This fly acts as a mechanical vector of pathogens, since it is associated with anthropic environments, and is frequently found on carcasses (Ferreira and Lacerda, 1993; Linhares, 1981). It is an insect of great medical and veterinary importance as it can cause myiasis in humans and in animals, principally in sheep. This fly is commonly known as the Australian sheep blowfly and can have an economic impact on the sheep industry (Stevens and Wall, 1996; Zumpt, 1965).
∗ Corresponding author at: Laboratório de Transmissores de Leishmanioses, Setor de Entomologia Médica e Forense, Instituto Oswaldo Cruz, Fundac¸ão Oswaldo Cruz, Av. Brasil, 4365—Manguinhos, Rio de Janeiro, RJ, Brazil. Tel.: +55 21 2562 1846/+55 2198495591. E-mail address: palomamm@ioc.fiocruz.br (P.M. Mendonc¸a). http://dx.doi.org/10.1016/j.actatropica.2014.04.007 0001-706X/© 2014 Elsevier B.V. All rights reserved.
© 2014 Elsevier B.V. All rights reserved.
Lucilia cuprina is considered an important forensic specie, since it has been frequently found associated with human and animal corpses according to many authors (Byrd and Castner, 2001; Early and Goff, 1986; Goff, 2000; Greenberg and Kunich, 2002; Smith, 1986). This fly is the dominant specie during the active decomposition stage, and is responsible for the removal of most of the tissue (Early and Goff, 1986). The blowflies (Diptera: Calliphoridae) are the first insects to arrive at a dead body and they readily oviposit on it (Smith, 1986). Therefore, knowledge of the length and activity of the larval stages of these flies can help determine the postmortem interval (Erzinclioglu, 1989). Most of these insects found on carcasses are the immature forms, and in order to help entomologists estimate the postmortem interval the correct identification of the immature forms is very important. The immature forms collected from corpses are commonly preserved in ethanol for later identification. However, there is a need to rear these larvae until adults in order to identify and analyze all the immature stages. The calliphorid larvae are very similar and some authors have described the first and third instar of L. cuprina using light microscopy focusing on the cephalopharyngeal skeleton, which is
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Fig. 1. Scanning electron micrographs of a first instar larva of Lucilia cuprina (Diptera: Calliphoridae). (A) Larval body with groups of spines located between the segments (arrows); the anterior (ae) and posterior ends (pe) of the larval body (×110). (B) Cephalic region with antennae (a), maxillary palps (mp) and bucal hooks (arrow) (x900). (C) Detail of spines between the first segment and thoracic region (×1100). (D) Anal segment with anal tubercles and posterior spiracles (arrows) (×500). (E) Posterior spiracles (arrow) (×1900).
the main structure visible using this technique (Sukontason et al., 2010; Szpila et al., 2013). However, light microscopy does not provide details of other characteristics that could have a diagnostic value (Liu and Greenberg, 1989; Mendonc¸a et al., 2010). Thus, the aim of this study was to describe and analyze the morphological characteristics of all larval instars and puparia of L. cuprina using scanning electron microscopy (SEM) in order to elucidate new diagnostic features of this specie.
and examined under a Jeol JSM 6390LV scanning electron microscope (Akishima, Tokyo, Japan). The SEM images were transferred directly to a computer. Puparia were not submitted to any kind of chemical fixation. Puparia were put under refrigeration for 5 min and then they were placed onto double-stick tape on metallic supports, coated with gold and examined under the same SEM. 3. Results
2. Material and methods 3.1. First larval instar The immature samples of L. cuprina used in this study were collected in Macapá, Amapá State, Brazil, using pig carcasses as bait, according to Barbosa et al. (2009). The colonies were reared and maintained at the Setor de Entomologia Médica e Forense, which is part of the Laboratório de Transmissores de Leishmanioses, Instituto Oswaldo Cruz, Fundac¸ão Oswaldo Cruz, in the state of Rio de Janeiro, as previously described (Queiroz and Milward-de-Azevedo, 1991). Insects were kept in cages at room temperature and supplied ad libitum with water and sugar. Bovine meat was used as a source of protein to stimulate oviposition and as food for the immature larvae. The third generation of the laboratory colony was used in this study. Ten specimens of each immature stage were analyzed. The terminology used in describing the morphology in this paper followed Margaritis (1985) and Mc Alpine et al. (1981). The three larval instar forms were washed several times in distilled water. Larvae were killed by placing them in hot water for 5 min and then they were fixed in 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer, pH 7.2, for 1 h and postfixed in 1% osmium tetroxide in the same buffer for 1 h at room temperature in the dark. Then these immature forms were washed with sodium cacodylate buffer three times for 10 min each. Afterward, they were dehydrated in an ascending series of acetone up to 100% and submitted to the critical point drying method, using superdry CO2 in a Balzers apparatus (Hayat, 1970). Specimens were placed on metallic supports, coated with a thin layer of gold (20–30 nm)
The larval body of L. cuprina is composed of 12 segments (one cephalic; three thoracic and eight abdominal) and has the vermiform shape typically found in muscoids (Fig. 1A). The anterior end, comprising the cephalic region, is pointed and the posterior end is blunt. The larval body is 3.76 ± 0.27 mm long and 0.199 ± 0.037 mm wide. The cephalic region is slightly bilobed and bears the sensorial structures: a pair of antennae and a pair of maxillary palps. Because of its localization, antennae are also known as dorsal organs and maxillary palps are known as terminal organs. The maxillary palps are formed by a complex of sensitive papillae. This cephalic region also includes the buccal hooks and oral cristae, but these structures are not well developed in the first instar (Fig. 1B). There is a band of spines between the cephalic region and the first thoracic segment. Two types of spines were observed: one type is flattened with tapered ends and the other is narrower with tapered tips, all of them are projected backward (Fig. 1C). The body tegument is smooth with the intersegmental line similar to those of the first thoracic segment. In this larval instar, the anterior spiracle is not visible. The posterior end or anal segment is covered with filiform spines and only the anal tubercles can be seen easily; the other tubercles are too small to be seen. The posterior spiracle is located at the top of an elevation and the single spiracular or peritrema opening is sustained by spiracular muscles (Fig. 1D and E).
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Fig. 2. Scanning electron micrographs of a second instar larva of Lucilia cuprina (Diptera: Calliphoridae). (A) Larval body with groups of spines located between the segments (arrows); the anterior (ae) and posterior ends (pe) of the larval body (×43). (B) Cephalic region with antennae (a), maxillary palp (mp), spines between the first thoracic segment and cephalic region (cs), oral cristae (oc), bucal hooks (bh) and anterior spiracles (as) (×180). (C) Details of antennae (a) and maxillary palps with papillae (mp) (×950). (D) Antennae (a), maxillary palps (mp) and spines between the segments (cs) (×700). (E) Details of anterior spiracles in a row (×850). (F) Posterior spiracles at posterior end (ps) and anal tubercles (at) (×170).
3.2. Second larval instar The body shape of the second larval instar is very similar to the first larval instar. The anterior end is pointed and the posterior end is blunt (Fig. 2A). The normal sizes of second instar larvae are 4.35 ± 0.27 mm in length and 0.624 ± 0.085 mm in width. The body tegument is smooth and the intersegmental spines are similar to those from the first thoracic segment and more visible than in the first instar. At the cephalic region, the bilobed shape is more pronounced and the antennae, maxillary palps and buccal hook are more developed than in the first instar (Fig. 2B and C). The band of spines from the first thoracic segment is flattened as found in the first instar, but the tips are not slender (Fig. 2D). The narrower spines were not observed in the second instar. The anterior spiracle, located in the first thoracic segment, is visible and well developed; it is composed of a row of 6–7 spiracular ramifications (Fig. 2E). The posterior end is still covered with filiform spines and the anal tubercles are more visible (Fig. 2F). A pair of posterior spiracles with two spiracular openings could be seen sustained by spiracular muscles (Fig. 2F). The peritreme is complete with an ecdysial scar (Fig. 2F).
posterior spiracle (Fig. 3F). Each posterior spiracle has three spiracular openings which are sustained by the spiracular muscles (Fig. 3G). The peritreme is still complete with an ecdysial scar (Fig. 3G). 3.4. Puparia In the puparia the anterior end is retracted and the posterior end is blunted (Fig. 4A). The puparia are 7.74 ± 0.67 mm in length and 2.18 ± 0.62 mm in width. The bands of spines limiting the segments are clearly visible. The spines are larger and flattened with slender tips like the third instar larvae. In the puparia of L. cuprina, there is a longitudinal ridge, which is a continuous line along the second and third thoracic segments (Fig. 4A). The anterior spiracle is located at the most anterior point of the cephalic region and is composed of 6–7 spiracular ramifications (Fig. 4B and C). At the end of the first abdominal, a tubular respiratory horn with longitudinal respiratory openings is visible (Fig. 4D). Each posterior spiracle, like the third instar larvae, has three spiracular openings with a clearly visible radial form and an opened peritreme without a scar (Fig. 4E). 4. Discussion
3.3. Third larval instar The third larval instar is very similar to the other two instars but all cephalic structures are fully developed (Fig. 3A and B). This larvae is 9.18 ± 0.19 mm long and 1.432 ± 0.7 mm wide. The band of spines from the first thoracic segment are flattened and the tips are very small (Fig. 3C). The intersegmental spines are large and flattened with slender tips and there are also some smaller spines with almost blunt tips (Fig. 3D). The anterior spiracle is composed of a row of 6–7 spiracular ramifications (Fig. 3E). The filiform spines in the anal segment are smaller than in the other two instars. The anal, ventral and dorsal tubercles are more visible and are important to protect the
The larval bodies of all muscoids are very similar. The vermiform shape found in L. cuprina is typical. This fly shares some characteristics with others blowflies of the same genus. In Latin America, the genus Lucilia blowfly is always collected rearing on carcasses, and in colder weather, it is one of the first to oviposit and rear on a dead body (Barreto et al., 2002; Carvalho et al., 2000; Ortloff et al., 2012; Salviano et al., 1996). Some authors have related the difficulties to identify the larvae using light microscopy (Oliveira et al., 2007; Queiroz et al., 1997), and the problems to rear these flies until adults in order to be able to use the taxonomic keys available. So, the use of scanning electron microscopy could provide diagnostic characteristics to help
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Fig. 3. Scanning electron micrographs of a third instar larva of Lucilia cuprina (Diptera: Calliphoridae). (A) Larval body with groups of spines located between the segments (arrows); the anterior (ae) and posterior ends (pe) of the larval body (×14). (B) Cephalic region with spines between the first segment and thoracic region (cs), antennae (a), maxillary palp (mp), oral cristae (oc) and bucal hooks (bh) (×170). (C) Detail of spines between the first segment and thoracic region (×400). (D) Detail of spines between the body segments (×280). (E) Details of anterior spiracles in a row (×550). (F) Anal segment with posterior spiracle (ps) dorsal tubercles (dt), ventral tubercles (vt) and anal tubercles (at) (×43). (G) Posterior spiracles with three spiracular openings (arrows) and ecdysial scar (s) (×160).
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Fig. 4. Scanning electron micrographs of a puparia of Lucilia cuprina (Diptera: Calliphoridae). (A) Puparia composed of groups of spines located between the segments (arrows); the anterior (ae) and posterior ends (pe) of the puparia; longitudinal ridge (dotted arrow); respiratory horn (circle) (×30). (B) Anterior region retracted (arrow) and anterior spiracle (ae) (×75). (C) Details of anterior spiracles in a row (×450). (D) Detail of spines between the body segments (s); respiratory horn (rh) (×450). (E) Posterior spiracles with three spiracular openings (arrows) and without an ecdysial scar (s) (×120).
entomologist identify the flies (Liu and Greenberg, 1989; Mendonc¸a et al., 2008, 2012a,b, 2013). Sandeman et al. (1987) described some characteristics of the larval instar of L. cuprina used from experimental infections with sheep. These authors focused on the mouth parts to verify if the first larval instar is able to damage sheep skin. As observed by these authors, the buccal hooks of the first instar larvae are very short but these parts are more developed on the second and third instar and are essential for feeding and locomotion. Apparently, there is no structural alteration among larvae reared on artificial diets and natural lesions. More recently, Szpila et al. (2013) analyzed six species belonging to the genus Lucilia in Europe, but they focused their work on the first instar larvae. The general morphology of the larvae is very similar to our observations. These authors used the spinulation of the abdominal segment A7 to distinguish between L. cuprina and L. sericata, as this is the main taxonomic question in Europe. Here, in Brazil, L. sericata is restricted to the south of the country and the
adult taxonomy is well established. However, due to the absence of studies comparing the larval morphology of the three species of the genus in Brazil, the doubts concerning the spinulation remain. In the L. cuprina collected in Amapá State, in the north of Brazil, the spinulation of segment A7 is incomplete, as related by Szpila et al. (2013). Although, the possibility of other arrangements could exist, since Wallman et al. (2005a,b) related the probable existence of cryptic species in L. cuprina. The number of spiracular ramifications is one of the most important characters to distinguish among dipterans species (Guimarães and Amorim, 2006). Sukontason et al. (2010) observed 4–7 openings at L. cuprina from Thailand using light microscopy. Sandeman et al. (1987) did not mention these structures. In the Brazilian L. cuprina species, the number of spiracular openings observed was 6 or 7. Greenberg and Singh (1995) related some morphological characteristics that could vary among dipteran species. These variations could reflect the alterations that have occurred to the species along
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