Larval morphology of the lesser housefly ... - Wiley Online Library

Medical and Veterinary Entomology (2012) 26, 70–82

doi: 10.1111/j.1365-2915.2011.00968.x

Larval morphology of the lesser housefly, Fannia canicularis 1

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A. G R Z Y W A C Z , T. P A P E and K. S Z P I L A 1 Department

of Animal Ecology, Institute of Ecology and Environmental Protection, Nicolaus Copernicus University, Toruń, Poland and 2 Department of Entomology, Natural History Museum of Denmark, Copenhagen, Denmark

Abstract. The morphology of all larval instars of Fannia canicularis (Linnaeus) (Diptera: Fanniidae) is documented using a combination of light and scanning electron microscopy. The following structures are documented for all instars: antennal complex; maxillary palpus; facial mask; cephaloskeleton; ventral organ; anterior spiracle; Keilin’s organ; posterior spiracle; fleshy processes, and anal pad. Structures reported for the first time for all instars include: two pairs of lateral prominences on the prothoracic segment; additional ventrolateral prominences on the second thoracic segment, and a papilla at the base of the posterior spiracle. Other structures reported for the first time are anterior spiracles in the first instar and a serrated tip on the mouthhook in the second instar. A trichoid sensillum on the posterior spiracular plate, representing a sensory organ otherwise unknown in the Calyptratae, is described in the second and third instars. Results are discussed and compared with existing knowledge on dipteran larval morphology. Key words. Fannia canicularis, larval instars, light microscopy, morphology,

scanning electron microscopy.

Introduction The Fanniidae represent a small dipteran family with worldwide distribution, the highest diversity of which is found in the Holarctic region (Carvalho et al., 2003). To date, about 285 species within the four genera of Fannia Robineau-Desvoidy, Euryomma Stein, Piezura Rondani and the Australian endemic Australofannia Pont have been described. Domínguez & RoigJuñent (2008) recently argued for the existence of a fifth, undescribed genus, endemic to New Zealand. For several decades the Fanniidae were classified as a subfamily within the Muscidae (e.g. Hennig, 1952; Chillcott, 1961, 1965; Ishijima, 1967; Huckett & Vockeroth, 1987) and this description can still be seen in the applied literature, especially in textbooks (Moon, 2002; Robinson, 2005; Byrd & Castner, 2009). Based on unique features of larval morphology, Roback (1951) raised the Fanniidae (from Fanniinae) to family rank in a move that has gained wide acceptance among more recent specialists (e.g. Pont, 1977, 2000; Rozkoˇsný et al., 1997; Carvalho et al.,

2003; Moores & Savage, 2005; Domínguez & Roig-Juñent, 2008). The Fanniidae’s status as a family is fully supported by recent molecular phylogenetic analyses (Kutty et al., 2008, 2010). McAlpine (1989) listed the following larval features as Fanniidae autapomorphies: a flattened body with usually branched fleshy processes, and posterior spiracles on raised processes. The form and arrangement of the fleshy processes characteristic of the Fanniidae are useful for species identification (Lyneborg, 1970; Duˇsek, 1971; Rozkoˇsný et al., 1997). Fannia canicularis (Linnaeus), known as the lesser (or little) housefly, is a eusynanthropic, cosmopolitan species (Rozkoˇsný et al., 1997; Bisby et al., 2009). It is common in the temperate regions of the world and less common in or even absent from tropical regions (Pont, 1977). Larvae of this species are associated with decomposing organic matter such as dung and rotting vegetables and fruits, but the species has also been bred in dead fungi, rotting wood, nests of insects (bumblebees, hornets, yellow jackets), nests and lairs of birds and mammals, decaying seaweed, and carcasses of insects, molluscs and vertebrates

Correspondence: Andrzej Grzywacz, Department of Animal Ecology, Institute of Ecology and Environmental Protection, Nicolaus Copernicus University, Gagarina 9, 87-100 Toruń, Poland. Tel.: +48 56 611 4469; Fax: +48 56 611 4443; E-mail: [email protected]

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Fannia larval morphology 71 (Ferrar, 1987; Rozkoˇsný et al., 1997). Of all species of Fanniidae, F. canicularis is most often involved in cases of myiasis (Zumpt, 1965; Ferrar, 1987). Larvae (Nuorteva et al., 1967; Benecke & Lessig, 2001; Benecke et al., 2004; Grassberger & Frank, 2004) and puparia (Vanin et al., 2007) of F. canicularis have been found on corpses during crime scene investigations and on animal carcasses used as models for decomposing human bodies. In such experiments larvae were even able to colonize buried bodies in favourable conditions (VanLaerhoven & Anderson, 1999). Descriptions of the morphology of F. canicularis larvae have been published by several authors. Tao (1927) described the first and second instars without providing any figures. Descriptions of the third instar with line drawings or photographs were provided by Hewitt (1912), Zimin (1948), Chillcott (1961), Ishijima (1967), Lyneborg (1970) and Holloway (1984). Additional figures representing the F. canicularis third instar, but without a description of larval morphology, were provided by Hennig (1952) and Huckett & Vockeroth (1987). Scanning electron microscopy (SEM) has been used previously in Fanniidae by Hinton (1981) to study egg morphology, by Couri (1992) to investigate the egg, third instar and puparium of Fannia pusio (Wiedemann), and by Al Gazi et al. (2004) to examine the eggs, third instars, puparia and adults of F. pusio and Fannia trimaculata (Stein). The aim of this study is to provide detailed morphological documentation of all larval instars of F. canicularis. For this purpose, both light microscopy and SEM were applied; the results are compared with previous descriptions of fanniid larvae and existing knowledge of the larvae of calyptrate flies. Materials and methods Larvae of F. canicularis were obtained by keeping wild-caught females in the laboratory until oviposition. Females were collected indoors on the campus of Nicolaus Copernicus University, Toruń, Poland, in August 2009, using an entomological net and were kept in 120-mL transparent plastic containers with wet sand in the bottom. Glucose and decomposed pork liver were used as food sources for adults and larvae, respectively. The cap of each container was perforated with small holes to allow for aeration. During the period after capture, the containers were examined twice per day to establish whether females had spontaneously oviposited. Some of the first instars were immediately preserved as described below. Remaining larvae were raised and preserved successively as older instars. Larvae of adequate age were transferred from the rearing containers to a Petri dish, killed by immersion in hot water (≈95 ◦ C), cleaned in distilled water with a fine brush and preserved in 70% ethanol. For light microscopy, larvae were slide-mounted in Hoyer’s medium. Cavity slides were used for first and second instars and for the cephaloskeleton of third instars. Larvae were photographed using a Nikon 8400 digital camera mounted on a Nikon Eclipse E200 microscope (Nikon Corp., Tokyo, Japan). Material prepared for SEM was dehydrated through 80.0%,

90.0% and 99.5% ethanol, processed in a Bal-Tec CPD 030 critical point drier, mounted on aluminium stubs with doublesided tape and sputter-coated with platinum for 140 s (20 nm of coating) using a JEOL JFC 2300HR high-resolution fine coater (JEOL Ltd, Tokyo, Japan). Images were taken with a JEOL scanning microscope (JSM-6335F; JEOL Ltd). Scale bars on SEM images are given in micrometres. Terminology follows Courtney et al. (2000) for general morphology and Lyneborg (1970), Holloway (1984) and Ferrar (1987) for family-specific structures with a few modifications proposed by Szpila & Pape (2005). We denote segments 1–3, 4–10 and 11 sensu Lyneborg (1970) as, respectively, T1–3 (thoracic segments), A1–7 (abdominal segments) and the anal division (the terminal part of the body posterior to A7 and probably composed of several segments). Particularly for the prominences and processes of the body wall that encircle most segments, we follow Lyneborg (1970) in using the positional terms ‘dorsomedian’ (DM), ‘dorsolateral’ (DL), ‘laterodorsal’ (LD), ‘lateroventral’ (LV), ‘ventrolateral’ (VL) and ‘ventromedian’ (VM). The processes of the anal division do not conform to the pattern of the regular segments and we follow Lyneborg (1970) in using the terms ‘lateral’ (L), ‘sublateral’ (SL) and ‘subapical’ (SA). Long fleshy structures covering the larval body are named ‘processes’. The term ‘prominences’ is reserved for small structures, usually with a papilla in the middle, which can be wart-like or surrounded by a ring of short projections.

Results The general morphology of the F. canicularis larval instars has been described previously in some detail, in particular by Hewitt (1912), Tao (1927) and Lyneborg (1970). The current study concentrates on features that were insufficiently described, wrongly interpreted or entirely omitted.

Pseudocephalon According to the position of the pseudocephalon (retracted, partly or fully extended), 11 or 12 body segments are visible (Figs 1A, 2A, 3A, 3D, 4A). The pseudocephalon is bilobate (Figs 1C, 2A, 3D) and the anterior of each lobe carries the antenna (Figs 1F, 2F, 3F), maxillary palpus (Figs 1D, 2E, 3E) and ventral organ (Figs 1E, 2H, 3G). The maxillary palpus consists of three sensilla coeloconica, three sensilla basiconica and one or more small additional sensilla, all in a tight cluster, as well as two additional sensilla coeloconica possibly of nonmaxillary origin (Figs 1D, 2E, 3E). The antennal complex includes a basal pore and a lateral pore with a sensillum (Figs 1F, 2F, 3F).

Cephaloskeleton The cephaloskeleton of the first instar significantly differs from those observed in second and third instars. The firstinstar cephaloskeleton is typical of the schizophoran type

© 2011 The Authors Medical and Veterinary Entomology © 2011 The Royal Entomological Society, Medical and Veterinary Entomology, 26, 70–82

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Fig. 1. First instar of Fannia canicularis. (A) Anterior end of body, dorsolateral view. (B) Respiratory aperture. (C) Pseudocephalon, ventral view. (D) Maxillary palpus. (E) Ventral organ. (F) Antennal complex. (G) Keilin’s organ on T1. AP, anterior process; DL, dorsolateral process; DM, dorsomedian process; LD, laterodorsal process; LV, lateroventral process; LP, lateral process; MH, mouthhook; NS1 [NS2], first [second] additional sensillum coeloconicum; RA, respiratory aperture; SB1 [SB2/SB3], first [second/third] sensillum basiconicum; SC1 [SC2/SC3], first [second/third] sensillum coeloconicum. © 2011 The Authors Medical and Veterinary Entomology © 2011 The Royal Entomological Society, Medical and Veterinary Entomology, 26, 70–82

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Fig. 2. Second instar of Fannia canicularis. (A) Anterior end of body, ventral view. (B) Anterior spiracle. (C) Posterior spiracle, dorsal view. (D) Second thoracic segment, ventral view. (E) Maxillary palpus. (F) Antennal complex. (G) Keilin’s organ on T2. (H) Ventral organ. AVL, additional ventrolateral prominence; KO, Keilin’s organ; LP, lateral process; LV, lateroventral process; MH, mouthhook; NS1 [NS2], first [second] additional sensillum coeloconicum; P, papilla; S, posterior spiracle plate sensillum; SB1 [SB1/SB2], first [second/third] sensillum basiconicum; SC1 [SC2/SC3], first [second/third] sensillum coeloconicum; VL, ventrolateral prominence; VMA, anterior ventromedian prominence; VMP, posterior ventromedian prominence. © 2011 The Authors Medical and Veterinary Entomology © 2011 The Royal Entomological Society, Medical and Veterinary Entomology, 26, 70–82

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Fig. 3. Third instar of Fannia canicularis. (A) Anterior end of body, lateral view. (B) Lateral base of anterior process. (C) Anterior spiracle. (D) Anterior end of body, ventrolateral view. (E) Maxillary palpus. (F) Antennal complex. (G) Ventral organ. (H) Keilin’s organ on T1. LP, lateral process; NS1 [NS2], first [second] additional sensillum coeloconicum; P, papilla; SB1 [SB2/SB3], first [second/third] sensillum basiconicum; SC1 [SC2/SC3], first [second/third] sensillum coeloconicum. © 2011 The Authors Medical and Veterinary Entomology © 2011 The Royal Entomological Society, Medical and Veterinary Entomology, 26, 70–82


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Fig. 4. Fannia canicularis larvae. (A) First instar, habitus, lateral view. (B) First instar, cephaloskeleton, lateral view. (C) First instar, cephaloskeleton, ventral view. (D) Second instar, facial mask, ventral view. (E) Second instar, cephaloskeleton, lateral view. (F) Third instar, cephaloskeleton, lateral view.

and consists of an unpaired labrum, paired mouthhooks, an unpaired intermediate sclerite, paired parastomal bars and paired vertical plates, each with ventral and dorsal cornua and connected by a dorsal bridge (Figs 4B, 4C, 5A). The entire cephaloskeleton is weakly sclerotized except for the anterior part of the labrum and the vertical plates (Fig. 4A–C). The mouthhook is shaped like a long, upwardly curved rod, with a slightly widened basal part carrying a lateral arm-like process (Fig. 5A). The mouthhook is apically equipped with a single tooth directed ventrally (the weak sclerotization makes the precise recognition of its shape difficult). The unpaired labrum is shaped like a broad knife blade (Figs 4B, 5A). The labrum is rigidly fused with the tips of the parastomal bars. Each parastomal bar is long and slender. The intermediate sclerite

is elongated and H-shaped (Fig. 5A). The crossbeam of the intermediate sclerite is perforated by an oval aperture. The vertical plate is slightly broader than the ventral cornu. The dorsal cornu is very short and about half as long as the ventral cornu. The cephaloskeletons of the second and third instars are very similar (Fig. 5B, C). The basal sclerite is long and consists of a very broad lateral plate, a rather short dorsal cornu and a longer ventral cornu that is very broad at its base. The dorsal bridge is massive and has characteristic perforations (Figs 4E, 4F, 5B, 5C). The parastomal bars are not developed. The intermediate sclerite is slightly elongated and H-shaped with a broad crossbeam (Fig. 5B). In the lateral view the crossbeam is visible as a distinct process on the ventral surface


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Fig. 5. Cephaloskeleton of Fannia canicularis larvae. (A) First instar, ventral and lateral view. (B) Second instar, ventral and lateral view. (C) Third instar, lateral view. DB, dorsal bridge; DC, dorsal cornua; DS, dental sclerite; IS, intermediate sclerite; LA, lateral arm; LB, labrum; LS, labial sclerite; MH, mouthhook; PB, parastomal bar; VC, ventral cornua; VP, vertical plate.

directed posteroventrally (Figs 4E, 4F, 5B, 5C). A paired sclerotization (labial sclerite?) is visible between the anterior arms of the intermediate sclerite. The paired mouthhooks are the anteriormost sclerites of the cephaloskeleton. The basal part of the mouthhook is robust and has a ventrolateral extension. The posterodorsal angle or corner of the mouthhook is drawn out into a distinct process. In the second instar, the middle part of each mouthhook is slender and straight, and the distal part is broader, slightly curved laterally, and equipped with several (about eight) teeth along the ventral margin. The connection between the slender part and the serrated apical part is difficult

to observe and the serrated part may be mistaken as a separate sclerite (Fig. 4E). In the third instar, the apical part of each mouthhook takes the form of a down-curved, pointed hook. Below the massive basal part of the mouthhooks are paired dental and labial sclerites.

Keilin’s organ Keilin’s organ consists of two trichoid sensilla (Figs 1G, 2G, 3H, 6C). The distances between pairs of pits with trichoid



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Fig. 6. First instar of Fannia canicularis. (A) Abdominal segments, dorsolateral view. (B) Anal division, lateral view. (C) Third thoracic segment, ventral view. (D) Ventromedian prominences on A1. (E) Posterior spiracle stalk, anterior view. (F) Anal pad, ventral view. (G) Ventrolateral prominence. (H) Posterior spiracle, dorsal view. KO, Keilin’s organ; L, lateral process on anal division; P, papilla; SA, subapical process on anal division; SL, sublateral process on anal division; ST, spiracular tuft; VL, ventrolateral prominence.

sensilla of Keilin’s organ are variable (Figs 1G, 6C): on T1 this distance decreases (in relative terms) from the first to the third instar, and is especially markedly reduced from the second

to the third instar, in which the pits become almost adjoined (Fig. 3H). The relative distance between pits is larger in T2–3 than in T1.


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Pattern of processes Two pairs of minute prominences with spine-like projections are present in all instars to the front of the anterior spiracle (Figs 1B, 2B, 3C). These prominences are barely visible in standard light microscopy. Two pairs of ventromedian prominences are present on T2–A7. On T2–3 these are arranged as an anterior and a posterior pair (Figs 2D, 7C, 7D), whereas on A1–7 they are arranged in a transverse line (Fig. 7E, F). One pair of additional ventromedian prominences is seen on A1–7 in the first and second instars and on A2–7 in the third instar (Fig. 7F). The ventromedian prominences of the first instar are simple wart-like structures without any visible central papilla or associated projections (Fig. 6D). In the second and third instars, the ventromedian prominences are equipped with spine-like projections surrounding the central papilla (Figs 2D, 7D–F), except for those on T2 in the third instar and the posterior ventromedian prominences on T2–3 in the second and third instars, which are simple papillae (Fig. 7C). The ventrolateral prominences present on T2–A7 have similar spine-like projections in all instars (Fig. 6G). On T2 between the lateroventral and ventrolateral prominences, in the middle of the segment, an additional ventrolateral prominence with spine-like projections can be seen (Fig. 2D); this is barely visible in standard light microscopy. No such additional structure was observed on any other segment. In the third instar, this additional structure is present as a papilla without surrounding projections, similar to the ventromedian prominences of this segment (Fig. 7C). The sublateral processes on the anal division are shorter than the lateral and subapical processes. The difference in length in the sublateral process is most distinct in the first instar (Fig. 6B).

Anterior and posterior spiracles The anterior spiracle in the first instar is present as a simple respiratory aperture (Fig. 1B), which is invisible in standard light microscopy. The anterior spiracles on subsequent instars are similar in shape, have about seven lobes, and are longer in the third instar. The posterior spiracles are raised on robust stalks, each of which has a papilla in the anterolateral position (Figs 2C, 6B, 6E). The first instar shows two slits, the second instar shows three slits on small baton-like projections and the third instar demonstrates each of the three slits on a small finger-like lobe. The plate of each of the posterior spiracles has four simple spiracular tufts in the first instar (Fig. 6H), one trichoid sensillum in the second instar (Fig. 2C), and a complex of two trichoid sensilla in the third instar (Fig. 7G, H).

Integumental sculpture The surface of the first instar lacks any trace of a polygonal pattern (Figs 1A, 6A–C); the body is covered with spines, which are more numerous on the anterior of each segment. Spines located to the posterior of each segment often have

branched tips. Ventrally, spines are limited to the anterior part of each segment (Fig. 6C) and the highest numbers of spines occur on T1–3. Dorsally and laterally, the middle and anterior parts carry simple spines, whereas the remaining part of each segment carries branched spines (Fig. 6A). In the second instar, the body is partly smooth, with a faint polygonal pattern, and is covered with a few wart-like prominences and small spines that are less numerous in comparison with those on the first instar. Ventrally, the spines are limited to the anterior part of each segment. The serrated tip of each mouthhook is exposed on the surface of the pseudocephalon and constitutes a part of the facial mask (Figs 2A, 5B). The third instar cuticle is pebbled with a polygonal pattern on the thorax (Fig. 3A) and shows an increasing number of wart-like projections to the posterior (Fig. 7A), some of which, especially those distributed laterally on the body, have pointed tips. T1 is without spines, whereas T2 and subsequent segments have simple spines located anterodorsally and anterolaterally (Fig. 7B, C). Segments A1–7 posterodorsally and posterolaterally show bands of spines and warts (Fig. 7A). The anterior processes and lateral parts of the body near their base are equipped with papillae (Fig. 3B).

Discussion Comparison with previous works The terminologies used by earlier authors differ from that proposed by Lyneborg (1970) and accepted by the present authors. Hewitt (1912), Tao (1927) and Ishijima (1967) numbered the segments from 1 (pseudocephalon) to 12 (anal division), and the ‘cephalic region’ described by Chillcott (1961) represents the combined pseudocephalon + T1–3, which explains why Chillcott (1961) denoted the anterior process on T1 as a ‘cephalic process’. Tao (1927) described five pairs of processes per segment in the first and second instars, but did not observe any ventromedian processes. Tao (1927) used the terms ‘lateral dorsal’ and ‘dorso-lateral’ interchangeably for laterodorsals and stated that ‘sessile branched appendages are situated near and slightly posterior to the base of the dorso-lateral appendages’ in a description that clearly refers to the dorsolateral prominences. Tao (1927) also mentioned that the laterodorsals ‘consist of 11 pairs of processes commencing on segment 3 and continued to the posterior end of the body’. Hewitt (1912) described a ‘latero-dorsal series of ten pairs of processes which commences on segment 3 and is continued to the posterior end of the body’. Assuming that the third segment sensu Hewitt (1912) and Tao (1927) is T2 and that the three pairs of processes on the anal division can be treated as laterodorsals (Tao, 1927; Lyneborg 1970), F. canicularis possesses 12 pairs of laterodorsals (nine on T2–A7 and three on the anal division), not 10 (Hewitt, 1912) or 11 (Tao, 1927). Zimin (1948) described four pairs of processes in the third instar, but did not describe dorsolaterals and did not differentiate ventromedians and ventrolaterals. Hewitt (1912), Zimin (1948) and Holloway (1984) noticed that the pseudocephalic segment is often withdrawn into the



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Fig. 7. Third instar of Fannia canicularis. (A) First and second abdominal segments, laterodorsal view. (B) Posterior end of body, dorsal view. (C) Second thoracic segment, ventrolateral view with position of papillae in place of additional ventrolateral process and ventromedian processes. (D) Third thoracic segment, ventral view. (E) First abdominal segment, ventral view. (F) Second abdominal segment, ventral view. (G) Posterior spiracle, dorsal view. (H) Posterior spiracle plate sensillum. AVL, additional ventrolateral process; AVM, additional ventromedian process; DL, dorsolateral process; DM, dorsomedian process; L, lateral process on anal division; LD, laterodorsal process; LV, lateroventral process; P, papilla; SL, sublateral process; SA, subapical process; VL, ventrolateral process; VM, ventromedian process.

prothoracic segment. Rozkoˇsný et al. (1997) and Pont (2000) mentioned that 11 visible segments can be found in larvae of Fanniidae, which can lead to the erroneous supposition that the pseudocephalon is reduced or is always retracted into the prothoracic segment. Retraction of the pseudocephalon

has been observed in response to factors such as mechanical stimulae in live larvae of all instars. In preserved material, configurations ranging from a fully extended to a fully retracted pseudocephalon were observed. Adams & Hall (2003) studied the influence of methods used for killing on the general


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shrinking of larvae and found significant differences in larval body length depending on water temperature and time of soaking. Similar influences may be suspected to affect the relative position of the pseudocephalon. Descriptions based on late third instars often stress the presence of 11 visible segments because the pseudocephalon is retracted during puparial formation in Fanniidae (Ferrar, 1987). It should be emphasized, as Hewitt (1912) did, that larvae of F. canicularis (and evidently other Fanniidae species) ‘consist of twelve segments, of which the first, or pseudocephalic segment, is often withdrawn’. In our opinion, the pseudocephalon should be included except in simple figures that show only speciesspecific patterns of processes. Of the three pairs of processes present on the anal division in F. canicularis, the sublateral is distinctly shortest (Fig. 7B). The length of this process is one of the characters upon which the identification of F. canicularis is based (Lyneborg, 1970; Rozkoˇsný et al., 1997). However, in Huckett & Vockeroth (1987; Fig. 50), the sublateral process appears to be at least as long as the lateral process. In Hennig (1952; Fig. 5), the sublateral processes are shown as only slightly shorter than the subapical pair. According to Hewitt (1912) and Lyneborg (1970), the sublateral process is localized closer to the lateral than to the subapical process. We observed such a position in the first instar (Fig. 6B), whereas, in the second and third instars, the sublateral process is positioned midway between the lateral and apical processes (Fig. 7B). This is in agreement with Zimin (1948; Fig. D3), Hennig (1952; Fig. 5) and Ishijima (1967; Fig. B). The posterior spiracles in the third instar of F. canicularis were described by Ishijima (1967) as ‘raised on [a] long stalk and with 3 or 4 branches’. However, describing the posterior spiracles of Fannia prisca Stein and Fannia scalaris (Fabricius), Ishijima (1967) mentioned only three branches. The posterior spiracle of third-instar Fanniidae, except in a few New Zealand species (Holloway, 1984), are known to have three more or less protruding lobes (Ferrar, 1987). The most probable explanation for such a description is that Ishijima (1967) misinterpreted the spiracular scar as a separate lobe (see Fig. 7G). Al Gazi et al. (2004; Fig. 5E, F) also mentioned four openings on the posterior spiracle of F. pusio and F. trimaculata, but used the term ‘openings’, not ‘branches’ or ‘lobes’. In Pont (2000; Figs 15, 16) the captions under the figures representing larvae of F. canicularis and fellow fanniid Piezura graminicola (Zetterstedt) (as Piezura boletorum Rondani) have probably been switched around in error. The figure representing F. canicularis refers to a Piezura and vice versa.

Anterior spiracles Generally, anterior spiracles are described as absent in firstinstar Cyclorrhapha (Ferrar, 1987). Couri (1992) described first-instar anterior spiracles in F. pusio as consisting of seven lobes. We consider that this opinion was based on a misinterpretation of a pharate second instar (i.e. an early second instar within the still-intact exoskeleton of a late first instar). Kitching (1976), studying first-instar larvae of several species of Calyptratae, reported the presence of a

simple, open orifice in a position at which the anterior spiracle might be expected. A similar feature has been found in species of the families Calliphoridae (Leite & Guevara, 1993), Muscidae (Grzywacz & Pape, unpublished data, 2010), Oestridae (de Filippis & Leite, 1997), Sarcophagidae (Cantrell, 1981; Leite & Lopes, 1989; Szpila, 2010; Szpila & Pape, unpublished data, 2010), Tephritidae (Elson-Harris, 1988) and Agromyzidae (Dempewolf, 2001). The respiratory aperture in F. canicularis would seem to resemble that described by Leite & Lopes (1989) in Peckia chrysostoma (Wiedemann) (Diptera: Sarcophagidae), although the wrinkles reported by the latter authors are possibly an artefact of their methods of preparation. A simple orifice without accessory structures has been reported in other species. We consider it very likely that a pair of very indistinct (but probably fully functional) spiracles will be found on T1 in other families of Cyclorrhapha. The common belief that anterior spiracles are absent in the first instar may have led students to ignore the possible presence of respiratory openings during SEM studies (e.g. Liu & Greenberg, 1989; Bonatto & Carvalho, 1996; Szpila & Pape, 2008; Draber-Mońko et al., 2009). Should a first-instar anterior spiracle be widespread in the Cyclorrhapha, methods of distinguishing first instars from subsequent instars should be modified as suggested by Kitching (1976), who described the anterior spiracles as ‘minute, not papillose, barely distinguishable using light microscopy’.

Serrated mouthhooks In his description of the morphology of second-instar F. canicularis, Tao (1927) did not report the presence of sclerotized structures on the facial mask, as were revealed here. We consider these sclerotized structures to be integral parts of the mouthhooks, although the connection with the remaining mouthhook is weakly sclerotized and difficult to observe in light microscopy. It should be mentioned that some authors (e.g. Skidmore, 1985) have treated such structures located apically on the cephaloskeleton as separate sclerites named ‘suprabuccal teeth’. No similar mouthhooks have been reported in any other Fanniidae (Lyneborg, 1970; Holloway, 1984; Couri, 1992).

Additional ventrolateral prominences Although they are positioned very closely to the lateroventral processes, we decided to name these prominences ‘additional ventrolaterals’ because of their similarity in shape to the prominences on the ventral surface. They are probably not serially homologous with any of the other processes as all six groups of processes are present on T2. It is more likely that these represent a seventh pair of sense organs that are fully developed and surrounded by spine-like projections only on T2 and present in the third instar only as a sensillum. Holloway (1984) also observed the structures we call ‘additional ventrolateral prominences’, but described them as lateroventrals, whereas lateroventrals sensu Lyneborg (1970) and previous authors were considered to be additional processes on T2. Given this contradictory information, and


Fannia larval morphology 81 based on the positions of lateroventrals sensu Holloway (1984) and sensu Lyneborg (1970)—the middle and anterior parts of the segment, respectively—we prefer to follow Lyneborg (1970) and treat the lateroventrals described by Holloway (1984) as additional ventrolateral prominences. Like Holloway (1984), we observed the presence of anterior and posterior ventromedians on third-instar T2–3, of which only the anterior ventromedians on T3 had surrounding projections. Similarly, in second-instar larvae, the anterior ventromedians and, in a few cases, even the posterior ventromedians, possess spine-like projections. To summarize earlier descriptions and the findings of recent data, all instars of F. canicularis have ventrolateral prominences on T2–A7, pairs of anterior and posterior ventromedians on T2–3 and two pairs of ventromedians on A1–7. An additional pair of prominences are found on A1–7 (on A2–7 in third instars). Posterior spiracle According to Ferrar (1987), spiracular tufts are found in Fanniidae only in the first instar; this claim is corroborated by the present study. The spiracular plates in second and third instars show one and two trichoid sensilla, respectively (Figs 2C, 7G, 7H). Holloway (1984) reported the presence of circular sensilla on the posterior spiracular plate in three undescribed New Zealand endemics, but Couri (1992) and Al Gazi et al. (2004) did not report any such sensilla in their SEM studies of two species of Neotropical Fannia. No additional information on the presence of such sensilla in other calyptrate species has been reported (Courtney et al., 2000). Such sensilla might be expected to occur in families in which the larvae have posterior spiracles that are similarly raised on stalks (e.g. in Platypezidae and Phoridae). However, no such sensilla were reported by Tkoˇc & Vaˇnhara (2008) in Lindneromyia hungarica Chandler (Diptera: Platypezidae) or by Boonchu et al. (2004) in Megaselia scalaris (Loew) (Diptera: Phoridae). Lateral prominences on T1 The small, lateral spine-like prominences present on T1 (Figs 1B, 2B, 3C) bear considerable similarity in structure and position to the laterodorsal and lateroventral processes present on successive segments. Using light microscopy, Holloway (1984) described the presence of one pair of minute processes consisting ‘of a ring of simple projections’ in F. canicularis. Despite describing two pairs of such minute lateral prominences on T1 in other New Zealand Fanniidae, Holloway (1984) did not observe a second pair of minute prominences in F. canicularis, as we observed below the anterior spiracle in the third instar. Acknowledgements We thank Dr A. C. Pont, (Oxford University Museum of Natural History, Oxford, U.K.) for comments on this manuscript and Professor M. S. Couri, (Museu Nacional,

Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil) for help with important literature.

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