Morphology of preimaginal stages of Calliphora vicina Robineau-Desvoidy, 1830 (Diptera, Calliphoridae): A comparative study

Forensic Science International 219 (2012) 228–243

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Morphology of preimaginal stages of Calliphora vicina Robineau-Desvoidy, 1830 (Diptera, Calliphoridae): A comparative study Nicola´s Ubero-Pascal a,b,*, Raquel Lo´pez-Esclapez a,b, Marı´a-Dolores Garcı´a a,b, Marı´a-Isabel Arnaldos a,b a b

Department of Zoology and Physical Anthropology, Faculty de Biology, University of Murcia, 30100 Murcia, Spain Unidad de Servicio de Entomologı´a Forense y Ana´lisis Microsco´pico de Evidencias, Servicio de Ciencias y Te´cnicas Forenses, Universidad de Murcia, Spain

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 November 2010 Received in revised form 29 December 2011 Accepted 3 January 2012 Available online 1 February 2012

A comparative morphological study of preimaginal stages (larvae and pupae) of Calliphora vicina RobineauDesvoidy, 1830 is presented. The entomological samples came from laboratory colonies bred under controlled environmental conditions (25 8C and 60% relative humidity). In this study, a recently published technique to clear Diptera larvae for light microscopy and a standard protocol for scanning electron microscopy were used. For the morphological comparison of larval instars I, II and III, and pupae of C. vicina, different larval regions (cephalic, thoracic and abdominal, including anal division), as well the internal chitinised cephalopharyngeal skeleton, were considered separately. Our results focus on showing the changes observed throughout development for the most important structures in the cephalic region (sensilla of maxillary palpus, antennae and oral ridges), the thoracic region (the first segment and its anterior spinose band) and in the anal division of the abdominal region (posterior spiracles and shape of the papillae). In addition, some morphological structures are described or pictured for the first time, such as the ventral organ and the anterior spiracle of larva I and the antenna sensilla, Keilin’s organ and wrinkled area of the anal division of all instars. The cephalopharyngeal skeleton is an important structure for the taxonomy of Diptera larvae in all instars, including Calliphoridae. Our observations in C. vicina indicate that an indepth review of the sclerite composition is needed. Pupae and larvae stages can only be compared by following the segmentary spinose bands and the anal segment, where the morphology of the posterior spiracles and papillae can be observed, in some cases despite the reduced condition of the latter. ß 2012 Elsevier Ireland Ltd. All rights reserved.

Keywords: Dipteran larvae Forensic applications Light microscopy Scanning electron microscopy Ultrastructure Calliphora vicina

Preimaginal stages of sarcosaprophagous Diptera are very interesting in forensic sciences from an applied point of view, because they provide relevant evidence for estimating the postmortem interval (e.g., Refs. [1–4]). Sometimes, the specific identification of preimaginal stages is difficult because some species are very similar morphologically, and they have not been studied in detail. Thus, in many cases, there are no good references to distinguish among them, especially in some geographical areas. Currently, the identification of preimaginal specimens requires breeding in the laboratory in order to obtain the adult stage. These adult stages have been studied in detail, and, for their identification, precise morphological parameters exist [5–9]. There is currently a large interest in studying preimaginal stages, and many articles have provided morphological descriptions of larvae and/or pupae, using mainly techniques of scanning electron microscopy (SEM). However, some of these publications are focussed solely in describing one of the larval stages, usually the

* Corresponding author. Tel.: +34 868 884960; fax: +34 868 883963. E-mail addresses: [email protected], [email protected] (N. Ubero-Pascal). 0379-0738/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2012.01.009

first or the third stages, or the pupae [10–23]. Only a few studies have dealt with the morphology of the complete preimaginal life cycle [24–31]. As mentioned above, the study of all the immature stages of sarcosaprophagous Diptera is of great interest, because the characteristic morphology of each one could provide specific features that will become essential for a proper identification. Historically, the studies of compared larval morphology of Diptera have been performed using light microscopy, especially when cephalopharyngeal skeleton was the focus of the study [12,32–34]. Hence, the main keys to distinguish Calliphorids, as well other Dipteran, have been performed based on this type of data [12,18,32,35,36]. Unfortunately, light microscopy is still the only technique available for many researchers in this kind of studies. We considered that morphological studies based on SEM techniques should be supplemented with light microscopy observations, because these techniques are complementary (e.g., Refs. [17,18,23,28,29,36,37]). While SEM provides accurate highresolution images of structures, light microscopy allows discrimination between structures with the same density to electrons, as cirri [22,23], or to observe internal structures, such as the cephalopharyngeal skeleton.

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Calliphora vicina Robineau-Desvoidy, 1830 (Calliphoridae) is a cosmopolitan species, occurring in urban and natural environments. It is highly associated with forensic cases involving dead humans and has also been related to different types of myiasis [38– 41]. Because of the above, many aspects of its biology have been considered and studied [10,32,42–44]. The morphology of its preimaginal stages has been thoroughly studied and described using mainly light microscopy techniques (e.g., Refs. [12,32]), but SEM studies are rare, and only deal with individual instar [10,12,23]. Besides this, larva II are practically undescribed from a morphological point of view, not only by SEM, but also by light microscopy, and some features of other instars are either not described, such as the wrinkled area of the anal division, or are not pictured, such as the ventral organ and anterior spiracle in larva I. Our work attempts to extend previous studies, not only by describing the morphological characteristics of every preimaginal stage of the life cycle of C. vicina using SEM and light microscopy techniques, but also by comparing the changes undergone by the main structures of the body. This article attempts to facilitate the specific identification of C. vicina in all preimaginal instars found in carrion, without the necessity to rear them in a laboratory. It also offers precise descriptions of morphological features already known, poorly characterised or not yet described, for systematic purposes. 1. Materials and methods Adults of C. vicina were collected in the Campus of Murcia University (Southeastern Spain) using a modified Schoenly trap [45]. Selected specimens were bred in the laboratory under controlled environmental conditions (25 8C and 60% relative humidity), using pig liver as substrates for egg-laying and larval food. Pupation substrate was sand surrounding the liver. All the immature specimens studied came from these sister colonies. All studied specimens, including adults, have been retained in the collections of the A´rea de Zoologı´a in the Departamento de Zoologı´a y Antropologı´a Fı´sica of Universidad de Murcia. A total of 120 specimens were studied: 40 specimens of larvae I and II, 20 specimens of larvae III and 20 specimens of pupae. They were selected, rinsed, euthanised in water near to boil (except pupae), and fixed in McDowell fixative solution (4% formalin, 1% glutaraldehyde mixture in cacodylate buffer solution, pH 7.4) [46], at 4 8C for 24 h. Specimens were rinsed again twice in sodium cacodylate + sucrose solution 0.1 M, dehydrated until absolute acetone in a gradient of increasing concentration of ethanol and ethanol–acetone solution (50% of each reagent) and dried using critical point method or by air-drying after hexamethyldisilizane treatment [47]. Dried specimens were mounted on SEM stubs with conductive adhesive tape, mounting them in different positions (dorsal, lateral and ventral views). For third instar larvae and pupae, larger than other instars, it was necessary to cut the specimens to study efficiently the fore and hind parts of the body. Specimens were coated with Au–Pd in a Polaron Bio Rad Sputter Coat and observed in a Jeol 6100 SEM. Pictures were directly obtained and digitised from the SEM. For light microscopy study, 10 specimens of larvae I and II and 5 specimens of larva III were chosen, euthanised in water near to boiling, fixed in ethanol 70% for 2 h, dehydrated until ethanol absolute and cleared in methyl salicylate (wintergreen oil) [48] for 1–3 h depending on the larval instar. Specimens were mounted in concave microscope slides using Canada Balsam as mounting medium. Microscope slides were observed using a Leica MZ 9.5 stereomicroscope with episcopic and diascopic illumination devices and a Nikon Eclipse i80 microscope with bright field and Nomarski interference contrast. Pictures from both microscopes were taken using a Nikon DS-Fi1 digital camera of 5-megapixel CCD and Nis-Elements software. To describe the morphological characteristics of larvae, cephalopharyngeal skeleton and pupae, the body divisions and the terminology given by Erzinc¸lioglu [32], Courtney et al. [49], Sukontason et al. [15] and Szpila et al. [23] were followed. 1.1. Abbreviations used in text and figures aI-VII, abdominal segments; ad, anal division; ae, additional spiracle structure; al, anterior labial sclerite; an, antenna; ans, antennal sensilla; a/ms, antennal or mandibular sensilla; ao, anal opening; ap, anal protuberance; as, anterior spiracle; asb-2, ventrolateral secondary anterior spinose band of tI; asb, anterior spinose band; b, buttom; bm, bubble membrane; br, basal ring; cd, cephalic depression; ci, cirri; cl, dephalic lobe; cf, cleft; cp, circular plate; cs, cephalopharyngeal skeleton; cw, creeping welt; db, dorsal bridge; dc; dorsal cornu; dm, antennal dome; es, ectostomal sclerite; fa, fan-shaped structure; fl, folding tegument; fm, facial mask; ig, intersegmental groove; is, intermediate sclerite; lb, labrum; la, labial arch; ll, labial lobe; lo, labial organ; ls, labrum supporting sclerite; mk, mouthhook; mk-3,

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infant mouthhook of instar III; mo, functional mouth opening; mp, maxillary palpus; mps, maxillary palpus sensilla; ms, medial spine; no, nose-like structure; os, oral sclerite; or, oral ridges; p1-7, posterior papillae; pap, papillae; pb, parastomal bar; pd, anal pads; pe, posterior spiracle; pl, posterior labial sclerite; pp, posterior projection; pr, peritreme; ps, pseudocephalon; ps-c, pseudocephalon collapsed; psb, posterior spinose band; pt, peristigmatic tuft; rh, respiratory horn; rp, rounded protuberance; rs, respiratory slit; sb1-3, basiconic sensilla of maxillary palpus; sc13, coeloconic sensilla of maxillary palpus; sd, dental sclerite; sp, spines; tI-III, thoracic segments; td, tiny digitations; vc, ventral cornua; vo, ventral organ; vop, ventral organ prominence; vos, ventral organ sensilla; vp, ventral plate; w, cornu window; wa, wrinkled area; ?, unidentified maxillary palpus sensilla.

2. Results and discussion The actual number of segments constituting the larval body of Cyclorrapha has been discussed, mainly those forming the cephalic region [49], but externally 12 can be identified (Figs. 4A and 5A) [11,16,19–21,23,25–27,29,32]. In Calliphoridae, the noticeable segments of the larvae could be grouped as pseudocephalon or cephalic region (first membranous segment, probably evolved from three embryonic segments), thorax (from second to fourth segments; they will be called tI, tII and tIII) and abdomen (from fifth to twelfth segments; they will be called aI to aVIII) [23,32,49], although the 12th segment is also known as the anal division (e.g., Refs. [32,49]: Fig. 7). The 12 segments can also be identified in Calliphoridae pupae, although the first and several parts of the anal division are collapsed [10,15,49]. Therefore, our results are presented regarding these different regions, except for those relating to the cephalopharyngeal skeleton and pupae. 2.1. Pseudocephalon morphological description and comparison Pseudocephalon is a highly specialised region of larval body; it contains several sensorial structures and, ventrally, the functional mouth opening. Morphologically, this region is very unique and its structures develop in a more complex way than in other segments. Pseudocephalon is bilobate anteriorly (Figs. 1A, 5C and 6B) or shows two latero-dorsal cephalic lobes. This feature is very clear in instar III because the lobes show a round shape and are separated from the rest of the pseudocephalon by a neck (Fig. 3A). In instars I and II the cephalic lobes are less conspicuous (Figs. 1A and 2A) and the bilobate characteristic is noticeable only dorsally; laterally and ventrally the cephalic lobes are delimited by the facial mask (Figs. 1A and 2A). The number of sense organs or, simply, cephalic structures used to describe this region, varies in the literature and, generally, depends on the larval instar. For instance, while Erzinc¸lioglu [32] notes five types of papillae to describe the cephalic region of Calliphoridae (antenna, oral papilla, maxillary palpus and two supramaxillary papillae), Courtney et al. [49] propose several other structures, such as labial lobe, cirri and oral ridges, some of which are described in detail by Szpila et al. [23] for C. vicina larvae I. Sometimes, the terms and structures considered in different articles do not agree completely, making their use difficult. Therefore, we attempt to describe the cephalic region in the three larval instars integrating and, where possible, discussing the different terminologies. Cephalic lobes are very interesting since each harbours two of the main sensorial structures, the antenna and the maxillary palpus. Their homologies to adult structures are not currently understood [32,49]. Antenna is disposed dorsally to maxillary palpus, which is placed in an antero-lateral position (Figs. 1A, 2A and 3A), and the relative distance between them is constant in all instars. Antenna is composed of two structures, a basal ring or socket and a distal dome (Fig. 1C). The relative size between dome and basal ring decreases from instar I to III (Figs. 1C, 2C and 3C). Antenna of instar I have a dome like a bullet and almost twice as long as the length of the basal ring (Fig. 1C), while in instars II and III the dome is conic-shaped and has a length equal to or less,

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Fig. 1. SEM morphology of pseudocephalon and anterior spinose band of first thoracic segment in the larval instar I. (A) Lateral view morphology; (B) detail of sensilla in maxillary palpus; (C) detail of antenna; (D) relative position of ventral organ in cephalic lobe; (E) detail of ventral organ; (F) detail of facial mask and labial lobe.

respectively, than the basal ring (Figs. 2C and 3C). A structure of interest, not described before in Calliphoridae, is a sensillum located in the external side of the edge of the basal ring. This structure could be related to the lateral receptor pore appearing in the groove between the two lobes of antenna in other species of Cyclorrapha [49]. In fact, it is also visible in other calliphorid species but it has never been discussed in the text (e.g., Ref. [28]: Fig. 16). Its morphology and, probably, structure change from instar I to III. It seems to be of basiconic-type in instar I, being located in a depression (Fig. 1C). In instar II, the margin of the basal ring is uniform in shape, but the sensillum is still visible, at least the tip, most of its length being included in the basal ring (Fig. 2C). In the instar III antenna (Fig. 3C), it is reduced to a simple hollow and is very difficult to identify. A transmission electron microscopy (TEM) analysis would be necessary to study the ultrastructure of this sensillum because it appears to be changing from basiconic in instar I to coeloconic in instar III, according to the description and classification of sensilla given by Zacharuk and Schields [50]. Maxillary palpus is structurally more complicated than antenna by the number and type of sensilla and their arrangement (Figs. 1B,

2B and 3B). Their complexity increases from instars I to III. According to Courtney et al. [49] the sensillae of maxillary palpus have different embryonic origins. It is more evident in instar I, where a sensillae cluster placed at the centre of a smooth round plate (maxillary origin) and two additional coeloconic sensilla (supramaxillary papillae following Erzinc¸lioglu [32]) in a dorsal position (antennal and/or mandibular origins) (Fig. 1B) can be noticed. In addition to the different embryonic origins, the additional sensillae are progressively integrated with the central cluster in instars II and III (Figs. 2B and 3C), and according to Szpila et al. [23] we consider that these sensillae should be treated morphologically as a part of the maxillary palpus. The cluster is usually composed of three coeloconic and three basiconic sensillae, although an additional basiconic sensilla has been detected in instars I and II (Figs. 1B and 2B), which is also shown in Szpila et al. (Ref. [23]: Fig. 4). Other Calliphorid species, as well as Sarcophagids and Muscids, show also one or two additional basiconic sensilla in the central cluster of maxillary palpus, although they have never been identified [17,18,21,22,25]. Moreover, Draber-Monko et al. [30] used a new way to call and number the sensillae in the central

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Fig. 2. SEM morphology of pseudocephalon and anterior spinose band of first thoracic segment in the larval instar II. (A) Lateral view morphology; (B) detail of sensilla in maxillary palpus; (C) detail of antenna; (D) detail of ventral organ; (F) detail of facial mask and labial lobe in lateral view.

cluster, for instance, they named elongated sensilla those widely known as basiconic sensilla number 3 [21,23,29]. In instar II, the maxillary palpus plate is folded in several ridges with different morphology (Fig. 2B): a circular ridge surrounding the sensillae cluster and several short ridges surrounding it; the additional sensilla closer to the cluster is placed in the centre of a triangular ridge and the second additional sensilla does not apparently change its shape and is not related to any ridge. In instar III, all the sensillae are surrounded by an annular ridge that forms a structure better defined and the folds appear to go deeper (Fig. 3B). However, the position and identity of sensillae in the maxillary palpus are constant, being easily identified in the different instars and allowing us to relate the different larval stages, as occurs in other calliphorid species [25,26,28], although it has not been previously reported. From a taxonomic point of view, Erzinc¸lioglu [32] considered these papillae not useful for specific diagnosis, at least using light microscopy. Evidently, the accuracy of light microscopy to discriminate this type of sensilla is not sufficient, especially in larvae I and II (Fig. 5E) but, with SEM, the ultrastructures of sensillae are excellent for taxonomic purposes in all instars (Figs. 1B, 2B and 3B). Moreover, we have noted that the pattern in maxillary palpus is not uniform among all the Calliphoridae species studied up to now. For instance, the number, morphology and topographic position of sensillae appear to differ between C. vicina and Chrysomya nigripes Aubertin, 1932 (e.g., Ref. [28]: Fig. 22) and several species of Pollenia Robineau-Desvoidy, 1830

(e.g., Ref. [17]: Fig. 8b or 13b) and Bellardia Robineau-Desvoidy, 1833 (e.g., Ref. [18]: Fig. 15 or 19). However, we agree with Szpila et al. [23], who argue that there is still not enough information at the SEM level and further studies will be necessary to decide on the taxonomic utility of maxillary palpus morphology. The remaining pseudocephalic structures (facial mask, ventral or oral organs and labial lobe) are more easily observed in ventral and lateral views (Figs. 1A, 2A and 3A). The facial mask is considered the complete area surrounding the functional mouth opening, and it is formed by two structures: oral ridges and cirri [23,49]. As in other Cyclorrapha, the oral ridges of C. vicina form the most conspicuous and extended structure (Figs. 2A and 3A), except in instar I (Fig. 1A), and are also distinguishable by light microscopy (Figs. 5E, 6F and 7C). They extend from the lateral margin of functional mouth opening to the lateral area of each side of pseudocephalon. In instar I, the oral ridge is an underdeveloped triangular-shaped area with no visible grooves inside (Fig. 1A and F). Nevertheless, this area seems to be well defined between two ridges perpendicular to the longitudinal larval body axis (Fig. 1F), as previously reported by Szpila et al. [23]. This structure is well developed in instars II and III and extended along a greater area than instar I, displaying many short grooves and ridges (Figs. 2E and 3D). They seem to radiate from the margin of the functional oral cavity (Fig. 3A) or the basis of the labial lobe (Fig. 2A). While in instar II the oral ridge conserves a triangular shape (Fig. 2A), in instar III this structure has lost this shape and is extended all along

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Fig. 3. SEM morphology of pseudocephalon and anterior spinose band of first thoracic segment in the larval instar III. (A) Lateral view morphology; (B) detail of sensilla in maxillary palpus; (C) detail of antenna; (D) detail of ventral organ and oral ridges; (F) detail of labial lobe in ventral view.

the pseudocephalon until the basis of the cephalic lobes (Fig. 3A). The fore margin of this structure surrounds each cephalic lobe at the back, allowing their differentiation in lateral-ventral view in instars I and II (Figs. 1A and 2A). Cirri are only visible in instar I as a row of small finger projections located in a ventral-medial position on each cephalic lobe, just above the oral ridge (Fig. 1A and F). Sometimes, as Szpila et al. [23] noted, cirri and the tip of the mouthhooks are very difficult to separate from each other by SEM (Fig. 1F), since this technique does not discriminate the degree of sclerotisation. As body spines, tips of cirri are often sclerotised allowing a correct differentiation from mouthhook by light microscopy, since it is a well-developed sclerite (Fig. 4B and C); Szpila et al. (Ref. [23]: Figs. 30–31) show these features adequately. In instars II and III we could not distinguish the cirri; their position seems to be occupied by a solid structure like a nose, with two openings for mouthhooks coming out (Fig. 3A). Courtney et al. [49] mention that the morphological variability of the facial mask is of great diagnostic value and varies between larval instars, as we have observed in C. vicina, between species of the same instar and between species of different families in Cyclorrapha. For instance, the oral ridge is not present in instar I of Pollenia species [17], while other Calliphorids have it well developed [25,26,28], and cirri are present like two bunches in necrophagous blowfly genera (Calliphora Robineau-Desvoidy, 1830, Phormia Meigen, 1826, Lucilia Robineau-Desvoidy, 1830, Hemipyrellia Townsend, 1918 and

Chrysomya Robineau-Desvoidy, 1830), although they are also present but substantially modified in parasitic genera such as Bellardia and Onesia Robineau-Desvoidy, 1863 [18]. As in other species of Calliphorids [23,28,49], first instar larvae of C. vicina present a ventral organ in an antero-ventral position of each cephalic lobe (Fig. 1D), as noted by Szpila et al. [23]. This structure is less conspicuous than those in instars II and III (Figs. 2A and 3A), although it can also be distinguished by light microscopy (Fig. 4E). Ventral organs are separated from the oral ridge in instar I and are composed of two or three sensillae and an anterior small papilla-like prominence (Fig. 1E). However, in instars II and III, the ventral organ is a clear structure located beneath each cephalic lobe just in the fore margin of the ventral area of oral ridge (Figs. 2A, 3A and D). Ventral organ in instar II is a globular protuberance located within a tegument depression (Fig. 2D), and presenting an oval-shaped notch in a medial position, in the margin of which some tiny digitations and a central prominence appear; the notch shows three sensillae (Fig. 2D). It is very similar to the ventral organ described in Chrysomya megacephala (Wiedemann, 1818) [25]. In instar III, ventral organ is a lentiform structure, like a button, that presents a slight fold in which some tiny digitations and the prominence appear clearly (Fig. 3D). Ventral organ are easily distinguishable in instars II and III using light microscopy although, as happens with other structures, the features are not visible (Fig. 5F).

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Fig. 4. Light microscopy morphology of internal and external structures in the larval instar I. (A) Larva habitus; (B) lateral view of the whole arrangement of cephalopharyngeal skeleton; (C) detail of the anterior (mouth-hooks) and medial parts (intermediate sclerites) of cephalopharyngeal skeleton in lateral view; (D) detail of anterior (mouth-hooks) and medial parts (intermediate sclerites) of cephalopharyngeal skeleton in ventral view; (E) detail of ventral organ; (F) morphological detail of anterior spiracle; (G) detail of the spinose band of thoracic segments in dorsal view; (H) detail of posterior spiracles.

Finally, the labial lobe appears posterior to the secondary mouth opening. In C. vicina it is visible in all instars (Figs. 1F, 2E and 3E) and, more or less, the structures composing it, as described by Courtney et al. [49], are easily distinguished – the properly named labial lobe and the labial organ located side by side. The labial lobe in instar I is formed of a single medial conic-shaped structure with the tip rounded (Fig. 1F). On each side there is a circular lobe, the labial organs, having a coeloconic sensilla in a central position (Fig. 1F), in the way that Szpila et al. [23] have noted. This pattern is retained in instars II and III, although in this last instar the labial lobe is triangular shaped with a large base (Fig. 3E) and, when the pseudocephalon is completely extended, this structure is articulated with a wide ventral plate, probably a submentum plate because the prementum is incorporated into a labial lobe according to Courtney et al. [49]. Concerning the labial lobe, we believe that this structure is present in instar I of all Caliphoridae larvae [17,23,49], despite the fact that it has been considered characteristic of instar II in some Chrysomya species [25,26]. The limit between the pseudocephalon and the first thoracic segment is unclear, at least in the dorsal area, and is difficult to decide what segment bears the first band of spines that appear in each instar of C. vicina (Figs. 1A, 2A and 3A). There are two observations that make it difficult to decide what segment is bearing the first spinose band: first, the presence of a ventrolateral fold behind the spinose band in all instars (Figs. 1A, 2A and 3A) and another, narrower, spinose band in a ventral position behind the tegument folder in instars II and III (Figs. 2A and 3A); second, the

first band of spines practically disappears when the pseudocephalon is retracted into the larval body. In all cases the cephalopharyngeal skeleton is extended internally in both segments (Figs. 4A, 5A and 6A), and also in segment tII, suggesting that the first spinose band belongs to both segments. However, because the publications addressing C. vicina larval morphology by light microscopy consider that the first spinose band is found on segment tI [12,23,32], we follow this terminology. Therefore, this feature of the larval body will be treated in the next section, with the other characteristics of the thoracic segments. 2.2. Morphological description and comparison of cephalopharyngeal skeleton Courtney et al. [49] discussed the confused status of cephalopharyngeal skeleton nomenclature for instar III and the difficulty in establishing sclerite homologies among species of Cyclorraphan Diptera. The question could be even more complicated if the arrangement of the cephalopharyngeal skeleton in all the larval instars is taken into account. In fact, if the terminology given in the works of Erzinglioglu [32] or Liu and Greenberg [12] is compared with more recent articles (e.g., Refs. [18,23,36]) in which the cephalopharyngeal skeleton of Calliphoridae is described, differences are found in both the nomenclature and the number of sclerites. Our observations of the cephalopharyngeal skeleton of instar I generally agree with the previous descriptions [12,23,32]. It is

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Fig. 5. Light microscopy morphology of internal and external structures in the larval instar II: (A) larva habitus; (B) lateral view of the whole arrangement of cephalopharyngeal skeleton; (C) ventral view of the whole arrangement of cephalopharyngeal skeleton; (D) detail of the anterior (mouth-hooks) and medial parts (intermediate sclerites) of cephalopharyngeal skeleton in lateral view; (E) detail of maxilary palpus; (F) detail of ventral organ; (G) detail of the anterior spiracle and spinose band of thoracic segments in lateral view; (H) detail of posterior spiracles.

composed (Fig. 4B and C) of a pair of mouthhooks, often with one or two teeth narrowest at the distal tip; paired ectostomal sclerites, sometimes slightly sclerotised and difficult to distinguish; unpaired beak-shaped labrum (Fig. 4C and D); unpaired intermediate sclerite, fork-shaped in ventral view (Fig. 4D); paired parastomal bars; and paired vertical plates each with ventral and dorsal cornua of same length. Both dorsal cornua are fused anteriorly forming a semicircular-shape dorsal bridge (Fig. 4B). A detailed analysis of intermedial sclerites of the cephalopharyngeal skeleton has allowed us to note a previously undescribed sclerite, in contact with the labrum sclerite and the posterior part of intermediate sclerite (Fig. 4C and D). This sclerite is normally poorly sclerotised and it may be that KOH treatment could depigment it, which would explain why it has not been reported before. The posterior tips of the labrum are articulated with the tip of two parastomal bars and supported below by the arms of intermediate sclerite (Fig. 4C). The newly described sclerite makes this joint stronger and helps the dorso-ventral movement of the labrum; we named it ‘labrum supporting sclerite’. The new technique used for clearing larvae of C. vicina allowed us to determine that the labrum sclerite and parastomal bar do not form a continuous unit (Fig. 4B and C), as usually represented in the literature [12,23,32]. On the contrary, it is formed at least by three sclerites and makes necessary a review of these structures in those species of Calliphoridae for which a description for larva I exists. The cephalopharyngeal skeletons of instars II and III are generally similar in the number and shape of their sclerites

(Figs. 5B, C, 6A and B). However, significant differences in both features can be described, such as the shape of mouthhooks and intermediate sclerites (Figs. 5D, 6C and D) and the presence of an unpaired oral sclerite in instar III (Fig. 6A and B). According to previous descriptions [12,32,36], in both instars we found paired mouthhooks, paired dental sclerites, unpaired intermediate sclerites, two units of labial sclerites (unpaired anterior and paired posterior), paired parastomal bar and a couple of vertical plates joined dorsally in a dorsal bridge and extended backwards in the paired dorsal and ventral cornua (Figs. 5B, C, 6A and B). The sickleshaped mouthhooks in instar II have a wider anterior than posterior section (Fig. 5B and D), while in instar III the anterior part is sharply curved and the posterior part is very broad (Fig. 6C and D); moreover, in instar II the posterior part has a well-developed posterior projection at the dorsal angle (Fig. 5B and D), which is absent in instar III (Fig. 6A and D). According to Courtney et al. [49], we have referred labial sclerites as two different processes in instars II and III, the first one is located between the posterior tips of the mouthhooks and the second one between the anterior arms of intermediate sclerite (Figs. 5C, 6B and C). Nevertheless, we think that these sclerites must be documented with different names, as proposed by Erzinc¸lioglu [32]. This author named them liguloid arch and hypostomal plate, respectively. However, we cannot propose these names because their origin is not clear [49]. Moreover, when comparing the relative position of sclerites, and the joints among them, in instars II and III with the cephalopharyngeal skeleton of

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Fig. 6. Light microscopy morphology of internal and external structures in the larval instar III. (A) Lateral view of the whole arrangement of cephalopharyngeal skeleton; (B) ventral view of the whole arrangement of cephalopharyngeal skeleton; (C) detail of the anterior (mouth-hooks) and medial parts (intermediate sclerites) of cephalopharyngeal skeleton in lateral view focusing dental sclerite; (D) detail of the anterior (mouth-hooks) and medial parts (intermediate sclerites) of cephalopharyngeal skeleton in lateral view focusing oral sclerite; (E) detail of the anterior and medial parts of cephalopharyngeal skeleton in ventral view; (F) detail of anterior spiracle; (G) detail of the spinose band of thoracic segments in dorso-lateral view; (H) detail of posterior spiracles.

larva I, our observations would be consistent with the use of a new terminology, but this task is beyond the scope of the present study. Dental sclerites are triangular-shaped and located close to the base of the posterior part of the mouthhooks. The relationship between these two sclerites is complex and in instar III the identification of dental sclerite may be incorrect in some previous descriptions. Erzinc¸lioglu [32] noted that in C. vicina ‘‘. . .dental sclerite usually slender and with comma shaped tail.’’ (Fig. 6D) and this shape is different in C. vomitoria (Linnaeus, 1758), although the real form of that sclerite is triangular shape (Fig. 6C); errors in descriptions are related to problems with the focus under light microscopy. When a picture of the general arrangement of the cephalopharyngeal skeleton in lateral view is required (Fig. 6A), the whole structure is focussed at the median line, which provides the shape of the lateral part of labial sclerite (Fig. 6D and E), not the shape of the dental sclerite (Fig. 6C). To know which sclerite is being observed, dental or labial, the clue is the accessory oral sclerite (aos). If the aos is focussed, then the visible sclerite below the mouthhook is the labial sclerite (Fig. 6A, D and E). The arms of the intermediate sclerite in instar II show a medial spine directed backwards (Fig. 5B and D), while in instar III that structure disappears (Fig. 6A and C). According to Erzinc¸lioglu [32], this feature is common in Calliphorids, but it is not represented in Liu and Greenberg [12]. The dorsal cornu is longer than the ventral cornu in both instars and this last sclerite shows also a characteristic small window [12,32,36]. Finally, the oral sclerite

is only present in instar III and in lateral view looks mace-shaped (Fig. 6A). Likely due to the clearing technique used, we have observed in ventral view that the oral sclerite is continued posteriorly by a lightly sclerotised arch, which defines the limits of the labium (Fig. 6B and E). 2.3. Morphological description and comparison of larval thorax in the three instars Brachyceran larvae can be apneustic, metapneustic or amphineustic, although the condition could be different depending not only on the species studied, but also on the larval instar [49]. In general, Cyclorrapha instar I larvae are metapneustic, while instars II and III are amphineustic [12,17,23,25,28,32,49]. However, Erzinc¸lioglu [32] and Sukontason et al. [25] have reported the presence of anterior spiracles in instar I in several species of Calliphoridae, such as Chrysomya rufifacies (Mcquart, 1843) and Phormia regina (Meigen, 1826), although this structure is very different morphologically to that of instars II and III and, probably, not functional. Moreover, Kitching [51] not only noted the presence of anterior spiracle in instar I in other species of calliphorids, such as Lucilia sericata (Meigen, 1826) and Chrysomya bezziana (Villeneuve, 1914), but also proposed that they are functional. According to Kitching [51] and Szpila et al. [23], C. vicina does not follow the usual morphology of Cyclorrapha as regards the anterior spiracle, which is present in all larval stages appearing

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Fig. 7. SEM morphology of thoracic and abdominal segments in the larval instar I. (A) Relative position of anterior spiracle in the first thoracic segment and anterior spinose band in lateral view; (B) detail of anterior spiracle; (C) detail of Keilin’s organ with trichoid sensilla; (D) spinose bands in abdominal segments; (E) detail of the spinose band of abdominal segments in dorso-lateral view; (F) detail of the spinose band of abdominal segments in ventral view.

on either side of tI segment (Figs. 7A, B, 8A and 9A). In instar I, the anterior spiracle is a simple short-cleft perpendicular to the longitudinal axis of the body on a circular plate (Fig. 6B), located in a dorso-lateral position near the posterior margin of the segment (Fig. 6A). This structure is also distinguishable by light microscopy, although its simple form makes observation very difficult (Fig. 4F). The anterior spiracle in instars II and III has a fan-shaped aspect as indicated by wide and flat protuberances bearing a row of respiratory papillae distally (Figs. 7A and 8A) [12,23,32]. The shape of papillae, resembling a horse-shoe, is not different from those described in other species of Calliphoridae [25,26,28]: a small round protuberance bearing in the tip an elliptic disc with a central respiratory slit which reaches the posterior margin of the disc (Figs. 7A and 8A). Anterior spiracles have the same fan-shape in instars II and III, but the number of papillae varies from eight (most frequent) to nine in instar II, and from 10 (most frequent) to 11 in instar III. These ranges agree with those described by Erzinc¸lioglu [32] for British specimens of C. vicina, although it was suggested that the range varies depending on the geographical provenance of specimens. This characteristic of the anterior spiracle is most useful for diagnosis since each species has a limited range of lobes [32] and these can be easily distinguished using light microscopy (Figs. 5G and 6F).

Ventrally, the thoracic segments present a pair of Keilin’s organs (Fig. 7C), a feature that has not been observed before in C. vicina. However, Courtney et al. [49] state that Keilin’s organs appear widespread in Diptera, which, for instance, in Cyclorrapha display a three hair-sensilla structure emerging from a pit of the tegument (e.g., Refs. [17,22,26–28,30]). According to Erzinc¸lioglu [32], the body of Calliphoridae larvae has creeping welts in each segment that are armed with spines surrounding the segment, forming a band or ring and which are very prominent in instars II and III (Figs. 7A, D, 8C and 9C). Spines are present in the anterior area from segment tI and in the posterior area from segment aII to the anal division. In some segments, the anterior and/or posterior bands may not be complete (Fig. 8C). According to previous descriptions given by light and SEM techniques, spines on the three thoracic segments of C. vicina are only present in the anterior area of each segment (Fig. 7A) [12,23,32]. The width of the first spine band around the segment is not uniform. Thus, while in instar I it is wider in the ventral area than in the dorsal area (Fig. 1A), in instar III it is the opposite (Fig. 3A). However, in instar II the width is more or less uniform in the entire perimeter (Fig. 2A). The spine morphology in instar I varies with the position in the band: the spines near the pseudocephalon are triangular and flattened in the dorsal area, whereas in the ventro-lateral area they are

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Fig. 8. SEM morphology of thoracic and abdominal segments in the larval instar III. (A) Detail of anterior spiracle; (B) detail of the spinose band of thoracic segments in dorsal view; (C) spinose bands in abdominal segments; (D) detail of the spinose band of abdominal segments in ventral view.

compressed, with a wide base and a curved tooth, like a rosebush spine (Fig. 1A). Spines of the following row are less strong; they have a wide base and a long and thin tooth, sometimes bifid, in the dorsal area, whereas in the latero-ventral area they are conical with a narrow base and a long tooth (Fig. 1A). Robust spines near the pseudocephalon also appear in instars II and III, but some differences may be noted. In instar II, dorsal spines are flattened with a wide base and short and single tooth, sometimes bifid (like

in Fig. 8B); the most posterior rows of spines in this area are conical-shaped, very short, robust and aligned. The lateroventral spines in instar II are also conical-shaped, robust, short and aligned near the pseudocephalon, whereas in posterior rows they maintain the conical shape, but the teeth are longer (Fig. 2A). In instar III, dorsal spines are more robust than in other instars. Near the pseudocephalon they are also flattened, the bases of contiguous units can be fused appearing as bifid spines with long and wide

Fig. 9. SEM morphology of thoracic and abdominal segments in the larval instar III. (A) Detail of anterior spiracle; (B) detail of the spinose band of thoracic segments in dorsal view; (C) spinose bands in abdominal segments; (D) detail of the spinose band of abdominal segments in dorsal view.

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Fig. 10. SEM morphology of anal division in the larval instar I. (A) Posterior view of anal division; (B) detail of posterior spiracle; (C) detail of wrinkled area; (D) details of posterior papillae and spines; (E) detail of anal protuberance.

teeth; more distally spines are grouped in rows of from three to five units and the bases also seem be fused (like in Fig. 9B). Lateroventrally, spines are conical-shaped (Fig. 3A). Finally, the second spinose band, in a ventrolateral position, in instars II and III is composed of strong and conical-shaped spines arranged in rows from four to six units (Figs. 2A and 3A). The distance between the two ventral bands could be of diagnostic value in some Calliphorid species, although this still remains to be determined [32]. The distribution and morphology of spines in segments tII and tIII are less complicated than tI, since the width of the spine band is more or less uniform around the segment (Fig. 7A). We have only observed morphological differences between dorsal and lateroventral spines, except in instar I. As indicated by Szpila et al. [23], we have also observed that the spines in bands of segments tII and tIII of instar I are conical, with a wide base and a pointed tip or tooth, and this shape is uniform around the band (Fig. 7A); no bifid teeth have been detected. On the other hand, dorsal spines in instars II and III are mainly flat and triangular-shaped, with some of them bifid (mostly in instar II (Fig. 8B)), and arranged in short rows disposed in an intercalated manner (Figs. 8B and 9B). The short rows are composed of three to five units in instar II and of three to eight in instar III. The most anterior rows may be conic-shaped, similar to those from the lateroventral area, but less strong (Fig. 9B). In these instars, lateroventral spines are conic-shaped with a strong base and a pointed tip, and generally are disposed

randomly, although in instar III they can be arranged in short rows of from three to five units. According to Erzinc¸lioglu [32], the morphology and degree of pigmentation of the spines (Figs. 4G, 5G and 6G) could be useful for taxonomic purposes in Calliphora species, that is, allowing discrimination between C. vicina and C. vomitoria in the third instar. Unfortunately, SEM techniques do not enable an assessment of the pigmentation. The similar morphology of dorsal spines in instars II and III indicated by this technique could be useful to distinguish also the second instar when spines of instar II of C. vomitoria are described by SEM microscopy. 2.4. Morphological description and comparison of larval abdomen in the three instars The abdominal segments of Calliphorids may show anterior and posterior spinose bands from aII to anal division, although they do not constitute a whole ring in all the segments, this characteristic being useful for a diagnostic study [12,23,32]. In general, our observations on spine distribution on abdominal segments agree with the descriptions given by Erzinc¸lioglu [32] and Szpila et al. [23] for each larval stage, but these descriptions do not fully agree with those given by Liu and Greenberg [12]. Thus, as reported by Erzinc¸lioglu [32], the spinose distribution on larval body could change depending on the geographical provenance of the specimen, as discussed above in relation to the number of lobes

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Fig. 11. SEM morphology of anal division in the larval instar II. (A) Posterior view of anal division; (B) detail of posterior spiracle; (C) detail of wrinkled area; (D) details of posterior papillae and spines; (E) detail of anal protuberance.

of the anterior spiracle. In whatever instar of C. vicina, the anterior spinose bands encircled the abdominal segments until segment aV (Figs. 7D and 8C). In segment aVI the band is interrupted dorsally, although some faint spines could appear; however, in segments aVII and the anal division spines do not appear dorsally (Fig. 9C). The posterior spinose bands appear from segments aII to aVII: these completely encircle the body from segment aIII to aVI, but is interrupted dorsally in aII (Fig. 8C), with the posterior area of anal division being different, as we will indicate below. Lateral posterior spines appear in all abdominal segments, including segment aI, although they are very faint (Figs. 7D, 8C and 9C). Anterior and posterior spinose bands are separated dorsolaterally by an intersegmental groove, which is very conspicuous in instars II and III (Figs. 8C and 9D). Spines are directed forward in the anterior spinose band, and directed away from the body in the posterior spinose band; this feature easily differentiates the bands (Figs. 7E, F and 8D). It has enabled recognition that, in instar III, the last segments show a branch differentiation of the anterior spinose band ventrally, which could either be Y-shaped (Fig. 9C), as Erzinc¸lioglu [32] reported, or form two separated branches. Spines in abdominal segments have the same shape all along both the anterior and posterior spinose bands. Instar I larvae have spines similar to those of segment tI close to pseudocephalon, in that they are compressed and with a curved tooth (Fig. 7E and F). In instar II, they are similar to instar I, but more robust and not

compressed (Fig. 8D); they can be organised in short rows or randomly. In hind segments of larvae II, spines can be conic shaped with the curved tooth; some small tubercles can appear widespread by the tegument (Fig. 8D). Larvae III have conic and robust spines with a curved tooth (Fig. 9D). They usually form short rows of three to eight units (Fig. 9D). The tegument between the spinose bands presents many tubercles or rounded protuberances (Fig. 12D and E). As with the pseudocephalon, the anal division in Cyclorrapha is a structurally complex area due to the fact that it is not clear from how many embryonic segments it is derived and because it has many structures with diverse functions [49]. According to several authors [12,23,25,26,28,32], these structures can be of taxonomic value in Calliphoridae, although in some genera or larval stages this is disputed [17]. In C. vicina, like other Calliphoridae, the anal division is dorsally obliquely truncated (Fig. 9C). The dorsal half is the spiracular field that carries some of the typical structures of Cyclorrapha, such as posterior papillae (or cones), the spinose band and posterior spiracles (Figs. 10A, 11A and 12A). The ventral half is projected ventrally forming the anal protuberance (stick-like anus), where the anal opening appears, a pair of lateral lobes (anal pads), and spines with particular morphology and arrangement (Figs. 10A, E, 11A, E, 12A and E). The anal protuberance is quite constant in the larval stages except in instar I, while the spiracular field shows a great variability (Figs. 10A, 11A and 12A).

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Fig. 12. SEM morphology of anal division in the larval instar III. (A) Posterior view of anal division; (B) detail of posterior spiracle; (C) detail of wrinkled area; (D) details of posterior papillae and spines; (E) detail of anal protuberance.

The spiracular field is organised as described by Erzinc¸lioglu [32] and Szpila et al. [23] (Figs. 10A, 11A and 12A); nevertheless, a detailed study shows some new or poorly described structures – seven pairs of posterior papillae of which six are distributed perimetrally and one is located towards the inside. The papillae morphology and development vary among the different instars. In instar I papillae are little developed, conic shaped, have a basiconic sensilla in the tip and are difficult to observe because they can be hidden by the spines, especially the perimetral ones (Fig. 10D). In instar II, perimetral papillae are more developed and easy to see because the spines are comparatively shorter. They keep the conic shape and the basiconic sensilla (Fig. 11D). Papillae p7 are proportionally less developed and can lose the basiconic sensilla. Finally, in instar III, the perimetral papillae are well developed and conic shaped, but some of them have lost the sensilla at the tip (Fig. 12D). In this instar, the globular papillae (p7) are little developed. Some authors consider the arrangement of posterior papillae, as well as the relative distance between some of them, as a taxonomic character [12,32]; indeed, the relative distance between papillae p1, p2 and p3 enables differentiation of C. vicina from C. vomitoria [32]. Surrounding the spiracular field and papillae p7 is the posterior spinose band of the anal division (Figs. 10A, 11A and 12A). In instar I the spines are long and slender, like a filament (Fig. 10C and D). In instar II they are a bit shorter relative to larval size, but similar in shape (Fig. 11C and D). In instar III they are robust, conic shaped

and short, similar to those of previous segments, but with a straight tooth instead of a curved tooth (Fig. 12D). Between the spines, this instar presents rounded tubercles similar to those of precedent segments (Figs. 9C, 12D and E). The middle area of the spiracular field is composed dorsally of a pair of posterior spiracles, transversally lined (Figs. 10A, 11A and 12A) and, ventrally, has an area that, in instar I, shows two big circular protuberances distally truncated and a bit depressed (Fig. 10C). In instars II and III it is composed of two corrugated tegument subareas with concentric lines of small spines (Figs. 11C and 12C). These protuberant structures also appear in other species in which instar I has been studied [23,25,28], but they have not yet received a particular name. Therefore, in order to provide uniformity in the terminology, we use the term ‘wrinkled area’, as suggested by Erzinc¸lioglu [32]. Each posterior spiracle is formed by the central spiracular disc (Figs. 4H, 5H and 6H), bearing the spiracular slits, surrounded by a chitinous ring, the peritreme [32]. This organisation is clearly visible in instars II and III of C. vicina (Figs. 11B and 12B). In instar I, however, the peritreme is not clearly defined (Fig. 10B); there are two slits on a circular protuberance, associated with four peristigmatic tufts, following Courtney et al. [49] (other authors refer to them differently: sun-ray [32], multibranched spiracular hairs [26]). The number and position of the peristigmatic tufts are constant in all instars, although in instar III they are more branched (Figs. 11B and 12B). This feature has been shown in other studies of Calliphoridae by SEM, but it has

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not been referred to in the text [25,26]. This supports the opinion of Szpila et al. [23] concerning the potential systematic utility of these characters, but further studies are needed. In instars II and III, there are two and three spiracular slits, respectively. They are well differentiated, with swollen margins, their ventral ends coalescing towards the spiracle ventral margin (Figs. 11A and 12A). Between both slits in instar II and the most intense two slits in instar III there is an intermediate structure lacking slits, but with a peristigmatic tuft dorsally located (Figs. 11B and 12B). In instars II and III the spiracular disc is surrounded by a corrugated area, somewhat swollen in instar II, forming the peritreme (Figs. 11B and 12B). Ventrally, the peritreme has a little rounded depression, the button or ecdysial scar, towards which the slits converge. Posterior spiracles are of taxonomic interest; for instance, the relation between the diameter of spiracle and the distance between both spiracles (spiracle distance factor, SDF [32]) enables discrimination of C. vicina and C. vomitoria. We have measured SDF for C. vicina and confirmed that it falls in the range of variation given by Erzinc¸lioglu [32]. The anal protuberance is visible in all instars of C. vicina, although its size and the definition of structures increase from instar I to III (Figs. 10E, 11E and 12E). In instar I, the anal

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protuberance is more or less globular as well as the lateral anal pads (Fig. 10E). In instars II and III, the anal protuberance is sticklike and the anal pads are conic shaped with a basiconic sensilla in the tip (Figs. 11E and 12E). The position of the anus is constant in all instars, which is an interesting feature for taxonomic purposes [32]. In contrast, spines change in shape and arrangement between instars I, II and III. In instar I they are triangular (Fig. 10E) and elongated. In instars II and III they are conic and robust and arranged in semicircular rows around the anus (Figs. 11E and 12E) [32]. 2.5. Morphological description and comparison of puparia and larva III body Calliphoridae puparia show most of the morphological characteristics of larval instar III [12,14,15,28,32] except that some structures are collapsed. The puparium of C. vicina has been described several times, applying some of the taxonomic characters used to describe the third larval stage, such as spine arrangement in segments, relative distance between posterior papillae p1, p2 and p3, and SDF [10,12,32]. In C. vicina, the puparium is oval shaped with a slightly truncated posterior edge. The pseudocephalon is completely

Fig. 13. Morphology of pupa. (A) Pseudocephalon and anterior area of the first thoracic segment collapsed; (B) detail of not functional anterior spiracle; (C) spines distribution in both anterior and posterior spinose bands; (D) detail of the respiratory horn showing traces of bubble membrane; (E) posterior view of anal division; (F) detail of not functional posterior spiracle.

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collapsed (Fig. 13A), and, as a consequence, no structure in this region can be distinguished. The anterior spiracles are visible and, if they are well preserved, it is possible to count the number of their lobes (Fig. 13B). The spinose bands patterns are visible in the body segments as well as the spine morphology (Fig. 13C). Pupae have an exclusive and characteristic structure, the paired respiratory horns (Fig. 13D), appearing dorsolaterally in the fifth segment. Their morphology is useful for taxonomic purposes [12,32]. In young puparia, the position of the horns is occupied by a bubble membrane that disappears when the respiratory horn is developed. Features of the bubble membrane in C. vicina are described by Liu and Greenberg [12]. The anal division retains traces of all the structures of larva III, although the posterior papilla and the anal protuberance are collapsed (Fig. 13E). The posterior spiracles are the unique structure defining the morphology of larva III and both the slits and the peritreme can be distinguished in the puparium, although the peristigmatic tufts are deteriorated (Fig. 13F). Because these structures are preserved, although deteriorated, it is possible to apply certain relative distance indices in order to identify the species [42]. 3. Conclusion The comparative study of the three larval instars of C. vicina has allowed understanding of the way by which some structures have morphologically evolved. Some structures have been differentiated or pictured here for the first time in C. vicina, such as the ventral organ and anterior spiracle in instar I, and the sensilla of the antenna, Keilin’s organ and the wrinkled area in all larval instars. Other structures change considerably during larval development, for example, the antennae, ventral organs, facial mask, spines of the anal division and the anal protuberance. In contrast, some structures remain more or less invariable in both morphology and position, the most representative being the cluster of sensillae of maxillary palpus, labial lobe, anterior spiracle, spine arrangement in thorax and abdomen and the arrangement of posterior papillae around spiracular field. Light microscopy has allowed us to confirm most of the previous descriptions of internal and external morphology given in the literature, although several differences or omissions have been noticed. The most significant results were obtained for instar I in which a new sclerite of the cephalopharyngeal skeleton was identified, the ‘labrum supporting sclerite’. This observation changes the general arrangement of cephalopharyngeal skeleton in its intermediate part and could be a reference to understand the arrangement of this structure in instars II and III, although an in-depth study of this topic is necessary. On the other hand, the identification of labial sclerites, sensu Courtney et al. [49], and the relationships among mouthhooks and dental sclerites suggest a previous misidentification of dental sclerites in instar III. Ultrastructural studies of all larval instars in Calliphoridae species are scarce, especially in the genus Calliphora. Thus, our observations show many characters that, when compared with those available at SEM of species of other Calliphoridae genera (i.e., Chrysomya and Pollenia), appear to be useful for taxonomy. We believe that the data presented here can be of great value for establishing the systematics of the group. In addition, the morphological characters described here will allow a detailed comparison with C. vomitoria when it is studied, since its geographical distribution overlaps with that of C. vicina. In these cases, although the third instars of both species are easily distinguishable at light microscopy techniques, there are issues regarding instars I and II. So, we hope to address this problem applying SEM techniques to instars I and II of C. vomitoria.

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