The antenna of horse stomach bot flies: morphology and phylogenetic implications (Oestridae, Gasterophilinae: Gasterophilus Leach)

Antennae are among the most elaborate sensory organs in adult flies, and they provide rich information for phylogenic studies. The antennae of five out of eight species of Gasterophilus Leach (G. haemorrhoidalis (Linnaeus), G. intestinalis (De Geer), G. nasalis (Linnaeus), G. nigricornis (Loew) and G. pecorum (Fabricius)), were examined using scanning electron microscopy. The general morphology, including distribution, type, size, and ultrastructure of antennal sensilla were presented, and the definition of auriculate sensilla and sensory pits were updated and clarified. Eighteen antennal characters were selected to construct the first species-level phylogeny of this genus. The monophyly of Gasterophilus was supported by the presence of coeloconic sensilla III on the antennal arista. The species-level cladogram showed G. pecorum branching off at the base, and the remaining species forming the topology (G. intestinalis+ (G. haemorrhoidalis+ (G. nasalis+ G. nigricornis))). Our research shows the importance of the antennal ultrastructure as a reliable source for phylogenetic analysis.

In this study, we conducted a comprehensive morphological comparison of five species of Gasterophilus, including the distribution, types, size and ultrastructure of antennal sensilla. We used these data to construct the first (partial) species-level phylogeny for Gasterophilus.

Materials and Methods
Morphological study. Adult male and female specimens of G. haemorrhoidalis (Linnaeus), G. intestinalis (De Geer), G. nasalis (Linnaeus), G. nigricornis (Loew) and G. pecorum (Fabricius) were captured during 2009-2011 in Kalamaili Ungulate Nature Reserve, Xinjiang, northwest China. The specimens were pinned and air-dried on site before being deposited in Beijing Forestry University. Material of the Palaearctic species G. inermis (Brauer), as well as of the two Afrotropical species G. meridionalis (Pillers & Evans) and G. ternicinctus Gedoelst, were not available for the semi-destructive sampling necessary for the study of antennal sensilla. For general morphology, antennae were studied using an Olympus SZX16 stereoscopic microscope. Series of photographs were taken using a Canon 500D digital camera (Canon, Inc., Tokyo, Japan) mounted on the stereoscopic microscope and superimposed using Helicon Focus (Helicon Soft Ltd, Kharkov, Ukraine) on a Windows 7 platform. All micrographs were processed in Adobe Photoshop CS5 (Adobe Systems, Inc., San Jose, CA, USA).
To prepare samples for scanning electron microscopy (SEM) documentation, the heads of exemplar specimens were excised and rinsed in PBS buffer (pH 7.0) to remove surface debris. For further cleaning, the antennal funiculus (including arista) was dissected from the remaining antenna, cleaned with detergent solution in an ultrasonic cleaner (50 °C, 15 min) and dehydrated in a graded ethanol series. The prepared samples were air-dried and mounted on aluminium stubs with double-sided adhesive tape and coated with gold before observing in a    Table 3. Morphometric data of antennal sensilla in Gasterophilus nasalis (Linnaeus) (mean ± SD). Au = auriculate sensilla; Ba I = basiconic sensilla I; Ba II = basiconic sensilla II; Cl = clavate sensilla; Co I = coeloconic sensilla I; Co III = coeloconic sensilla III; Tr = trichoid sensilla I; F = female; M = male; − = undetermined.
HITACHI S34Q scanning electron microscope (Hitachi Corp., Tokyo, Japan) at the Microscopy Core Facility, Biological Technology Center, Beijing Forestry University (Beijing, China). The length, basal diameter, distal dilation diameter, and distribution of antennal sensilla were measured. As shown by Zhang et al. 12 , the types of antennal sensilla in male G. nigricornis are highly similar to those of the female, so for this species only the distribution of sensilla on the female antenna is given in this paper. The terminology follows Zhang et al. 12-16 . Phylogenetic analysis. Eighteen characters (13 binary, 5 multistate) were included in the analysis, which covered characters from antennal scape, pedicel, funiculus (including arista) and sensilla (Appendix 1). The matrix (Appendix 2) was compiled using MESQUITE 1.05 32 . No data are available on the antennal ultrastructure for the genus Gyrostigma Brauer, which is the most probable sister-group to Gasterophilus 33 , and no specimens are available for the semi-destructive sampling required for proper SEM-documentation. We chose Hypoderma lineatum (Villers) (Hypodermatinae) and Rhinoestrus purpureus (Brauer) (Oestrinae) as outgroup representatives, as the antennal ultrastructure of these two species has been extensively documented by Liu et al. 9 and Li et al. 10 , respectively. For proper rooting of the cladogram, we used Lucilia sericata (Meigen) (Calliphoridae) as a more distant outgroup whose Antennal ultramorphology was reported by Zhang et al. 15 . Phylogenetic analyses were performed under the parsimony criterion with the program TNT 1.1 34 . All multistate characters were considered to have their states forming linear transformation series as indicated under the relevant character following the description of character states. They were treated as either additive (= ordered) or non-additive as given in the discussion. Exhaustive searches using implicit enumeration were conducted under equal weights (= unweighted) and with implied weighting 35 under a range of k-values as given in the discussion. Branches were collapsed if the maximum branch length was zero.
Branch support for individual nodes was estimated by Bremer's decay index 35,36 . Suboptimal trees allowing for five extra steps were produced in TNT to calculate absolute Bremer support values. The scape, the first and shortest segment of the antenna, has a smooth cuticular surface without microtrichia. The pedicel (Figs 1A-C, 2A-C, 3A-C and 4A-C), the second segment, is flattened and disc-like and is covered with microtrichia on the surface. In G. pecorum, the pedicel is elongated and is the longest segment ( Fig. 1A-C). Mechanoreceptors (Figs 1C,D, 2C-E, 3C,D and 4C,D) are distributed both on the scape and pedicel. These are short, straight setae or bristles of varying length and with the usual longitudinal grooves on the cuticular surface. The pedicellar button (PB) (Fig. 5) is a button-like structure situated on the articular surface near the pedicellar cleft, and it consists of a circular dome centrally and a slightly convex (G. intestinalis and G. pecorum) or distinctly ridgy (G. haemorrhoidalis, G. nasalis, G. nigricornis) ring peripherally (Fig. 5). Gasterophilus pecorum has two buttons on the pedicel (Fig. 5A), whereas there is only one in the other four species.
The funiculus is the most conspicuous segment of the antenna, housing a variety of sensilla. It is roughly pyriform and can be divided into three aspects: the anterodorsal surface (Ad), the dorsolateral surface (with the arista)  (Figs 8 and 9), clavate sensilla (Fig. 10) and auriculate sensilla (Fig. 11A-C). The length, basal diameter, and distal dilation diameter of these sensilla for both males and females are summarized in Tables 1, 2, 3   Coeloconic sensilla. Coeloconic sensilla (Co) are the shortest of the sensilla, and are located in sunken cavities (Figs 8 and 9; Tables 1, 2, 3 and 4). Three subtypes (Co I, II, III) of Co can be distinguished: Co I and Co II are distributed on the mid-proximal region of the anterodorsal and the posteroventral surface of the funiculus, whereas Co III is found only on the arista. Co I (Figs 8A,E and 9A,D) is a short peg with deep, longitudinal grooves over the distal 1/3-1/4 of the sensillum, and it is found in all five species studied. Co II (Fig. 8B) is a relatively long peg with an irregular pattern on the terminal part. This type is found only in G. pecorum. Co III is usually short and smooth (Figs 8C,D,F,G and 9B,C,E,F), except in G. nasalis, in which it is approximately triangular in shape and with grooves ( Fig. 8E-G), and found exclusively at the base of the distal aristal segment. In G. intestinalis and G. pecorum, Co III are clustered in shallow depressions, while in G. haemorrhoidalis, G. nasalis, and G. nigricornis, they are distributed singly (Figs 8 and 9).   Clavate sensilla. The clavate sensilla (Cl) are characterised by a subapical dilation or swelling, giving them a club-like or spatulate appearance. This type of sensillum is seated in a superficial cavity (Fig. 10) and can only be observed on the most proximal region of the funiculus (Figs 12, 13, 14, 15 and 16). In G. nasalis (Fig. 10B) and G. nigricornis, each clavate sensillum has a short, tapering tip, while in the other species of Gasterophilus, the Cl is more abruptly tapered with the tip broadly rounded (Fig. 10A) or angulated (Fig. 10C,D). Additionally, clavate sensilla are distributed either singularly on the surface (Fig. 10A) or clustered in pits (Fig. 10B).
Auriculate sensilla. In Gasterophilus spp., the auriculate sensilla (Au) are gradually tapered from the base, with a length-width ratio < 3 (Tables 1, 2, 3 and 4), somewhat resembling the ear of a rabbit (Fig. 11A-C). Au are distributed either singly on the surface or clustered in sensory pits. Auriculate sensilla were first discovered by Setzu et al. 37 in Protophormia terraenovae (Robineau-Desvoidy) (Calliphoridae) and described as ear-or spoon-like formations presenting a concavely indented or sunken surface distally. In this study, we observed this type of sensilla in four species (G. haemorrhoidalis, G. intestinalis, G. nasalis and G. nigricornis). It should be noted that distinguishing antennal sensilla solely by their cuticular surface concavity can be misleading, since several sensilla that we observed (e.g., clavate sensilla) had partly sunken cuticular surfaces. They were situated amongst morphologically similar sensilla without any sign of concavity. Shrinkage and deflation happen occasionally during the dehydration process for scanning electron microscopy, which may explain this inconsistency 38,39 . Sensory pit. The sensory pit is a cave-like depression in the funicular surface that contains several sensilla of the same type (Figs 3E, 7C, 9A, 10B, 11C, 12, 13, 14, 15, 16 and 17). All Gasterophilus spp. have the funiculus equipped with numerous sensory pits.
The concept of sensory (or 'olfactory') pits needs clarification, as any depression on the cuticle in connection with sensilla are sometimes referred to as a sensory pit 37,40 . McAlpine 19 differentiated between "simple pits" as opposed to the "deep, sac-like invagination of the cuticle of segment 3 containing several trichoid sensilla and opening to the exterior by a relatively small pore", which Lowne 41 termed the 'sacculus' . Similarly, Zhang et al. 14 separated the 'sensory pit' as a single-chambered invagination containing a cluster of sensilla from the 'sacculus' , which was defined as a multi-chambered invagination stretching into the cavity of the antennal funiculus, and often with a complement of different types of sensilla. We propose that the term 'sensory pit' should be used to describe a cluster of sensilla located inside a saucer-or bowl-like depression of the cuticular surface with at most one third of their length emerging above the surrounding surface. A fringe of microtrichia is usually found around the edge of a sensory pit. Pezzi et al. 42 used the term 'olfactory pit' , but as antennal sensilla may have other functions, e.g., hygro-or thermoreception 43 , we prefer the broader term 'sensory pit' .
Phylogeny. Analysing the matrix under equal weights and with all characters treated as non-additive (i.e., unordered) yielded two most parsimonious trees. The strict consensus of the two trees gave a sister-group relationship between the clade Hypodermatinae + Oestrinae and a largely unresolved Gasterophilus (Fig. 18). Analysing the data as non-additive under implied weights resulted in a fully resolved tree for all values of k ≥ 1, which differs from the equally weighted analysis in the topology of Gasterophilus, with G. pecorum as sister taxon to all other species of Gasterophilus (Fig. 19). An identical tree is obtained in analyses with the multistate characters treated as additive, both in the equally weighted analysis and in analyses with implied weighting and for all k-values (i.e., k ≥ 1). Bremer supports for every node of the minimum-length cladogram are given in Fig. 19.
The genus Gasterophilus is supported as a monophyletic group (Fig. 19) based on two synapomorphies: both flat and hair-like microtrichia on antennal funiculus (C8: 1) and the arista with coeloconic sensilla III (C11: 0) (Figs 8C,D,F,G and 9B,C,E,F). A great diversity of antennal sensilla types and locations are evident within this small genus, compared with 3-6 types of antennal sensilla arranged mostly on the funicular surface in other flies 8,14,15,[43][44][45][46] . All species of Gasterophilus have seven types of sensilla, except for G. pecorum having only six types. These cover nearly all types of sensilla (e.g., plaques are not detected) described in cyclorrhaphan flies. Some sensilla can be divided into several subtypes, making a strict calculation of the total number of sensillar types in this genus potentially misleading. Besides, large numbers of sensilla are located in numerous sensory pits and so are more difficult to study. There is increasing evidence that specific types of antennal sensilla have specific      Within Gasterophilus, G. pecorum branches off at the base, emerging with four autapomorphies: the microtrichia are extensive on the outer side but absent on the inner side of the pedicel (C0: 2) (Fig. 1C), length-width ratio of antennal pedicel more than 0.8 (C1: 2), two antennal pedicel buttons (C3: 2) (13A), and two subtypes of coeloconic sensilla on funiculus (C10: 1) (Fig. 8A,B), and with four homoplasious character states: the pedicel partly enveloping the funiculus (C2: 1), the slim mechanoreceptor (C7: 0) with twisting grooves (C6: 1) but no socket (C5: 1) (Fig. 1D).
Gasterophilus pecorum has one more subtype of Co than all the other species included in the present analysis (C10: 1). Co are sensitive to many olfactory cues 48,[50][51][52][53][54] instead of being only hygro-or thermo-sensitive as speculated previously [55][56][57] . Further behavioural and electrophysiological studies are required to understand why G. pecorum has acquired more types of antennal coeloconic sensilla than other bot flies, but it is noteworthy that G. pecorum will deposit eggs at a distance from its host rather than directly on the host 23 , which is probably a derived behaviour that may require additional sensory input to monitor host location.
No prior study has specifically investigated a species-level phylogeny for Gasterophilus. Otranto et al. 58 analysed partial sequence data from the mitochondrial COI gene and the ribosomal genes 16S and 28S in a study of the "differentiation and phylogenesis" of five species of Gasterophilus. Unrooted Maximum Likelihood "phylograms" were given based on each gene, but no explicit phylogeny was presented. Rooting their three phylograms (Fig. 20) will generate phylogenies strongly conflicting with the present study (Fig. 19), which may be caused by the sparcity of molecular data. Otranto et al. 58 also suggested that G. haemorrhoidalis and G. intestinalis could be morphotypes of the same species based on high genetic similarity, in particular of the 28S gene (Fig. 20A). They even considered the main site of development of the third instar larva as sufficiently similar to support conspecificity in spite of known differences 31 . This view did not consider significant evidence that G. haemorrhoidalis and G. intestinalis are biologically and morphologically quite distinct in several features like oviposition site, oviposition behaviour, details of first instar larva (especially the cephaloskeleton) and adult male and female morphology (for an exquisite treatment see Grunin 31 , which incidentally was not cited by Otranto et al. 58 ). In the present study, we found significant differences in the antennal morphology of these two species (Figs 14 and 15), such as a distinctly ridgy ring of the pedicellar button and singly distributed aristal Co III in G. haemorrhoidalis, while G. intestinalis has the alternative state for both these characters (see the results and list of characters in Appendix 1). Actually, the existing similarities between the two species may relate entirely to their shared ancestry, i.e., be symplesiomorphic, because G. intestinalis is cladistically subordinate to G. haemorrhoidalis, with the latter being more closely related to G. nigricornis and G. nasalis.