First instar larvae of endemic Australian Miltogramminae (Diptera: Sarcophagidae)

The first instar larva of a species of the Australian endemic genus Aenigmetopia Malloch is described for the first time, along with the first instar larvae of three other Australian species representing the genera Amobia Robineau-Desvoidy and Protomiltogramma Townsend. Larval morphology was analysed using a combination of light microscopy, confocal laser scanning microscopy and scanning electron microscopy. The following morphological structures are documented: pseudocephalon, antennal complex, maxillary palpus, facial mask, modifications of thoracic and abdominal segments, anal region, spiracular field, posterior spiracles and details of the cephaloskeleton. Substantial morphological differences are observed between the three genera, most notably in the labrum and mouthhooks of the cephaloskeleton, sensory organs of the pseudocephalon, spinulation, sculpture of the integument and form of the spiracular field. The first instar larval morphology of Aenigmetopia amissa Johnston, Wallman, Szpila & Pape corroborates the close phylogenetic affinity of Aenigmetopia Malloch with Metopia Meigen, inferred from recent molecular analysis. The larval morphology of Amobia auriceps (Baranov), Protomiltogramma cincta Townsend and Protomiltogramma plebeia Malloch is mostly congruent with the morphology of Palaearctic representatives of both genera.

Thoracic segments. Except for t1, anterior spinose bands difficult to differentiate from warts of interband surfaces, ventral surface of segments smooth without spines or warts ( Fig. 2A,B).

Discussion
The first molecular-based reconstruction of the phylogeny of the Miltogramminae enabled a formal testing of the hypotheses for the evolution of the larval morphology and feeding strategies of this taxon 1 . The ancestor of the Miltogramminae was likely a saprophage specialised for utilising vertebrate carrion buried in the ground, as highlighted by the combination of its large pseudocephalic sensory organs and well-developed cuticular sculpture, along with retention of a plesiomorphic dorsal bridge in the cephaloskeleton 1 . Larvae of Australian species, including those of the endemic Aenigmetopia, share synapomorphies such as the loss of a dorsal bridge and hair-like spines around the spiracular field with other kleptoparasitic Miltogramminae. This evidence supports their position within the 'higher' miltogrammines sensu Piwczyński et al. 1 .
While the morphology of first instars of Am. auriceps, P. cincta and P. plebeia is generally similar to that of their Palaearctic relatives, the first instar of Ae. amissa appears to be the most intriguing. Hypotheses about the   9,12 presented molecular data in support of a sister-group relationship between Aenigmetopia and a subset of Metopia, rendering the latter genus paraphyletic. While this conflicts with the traditional morphological circumscription of Metopia 6 , it may be considered to constitute the best-supported hypothesis for the phylogenetic position of Aenigmetopia to date, even if the support is low (BS = 60, PP = 0.84) 12 . An interesting similarity is observed between Ae. amissa and Senotainia conica (Fallén) 2 , which share the following character states: (1) antennal complex with high basal ring, (2) club-like first sensillum basiconicum situated close to the central cluster of sensilla, (3) ventral organ sensilla placed on a small fleshy lobe, (4) oral ridges present, (5) mouthhook with serrated tip, and (6) cuticular sculpture in the form of regular ridges. However, most of these character states cannot be considered to provide a strong phylogenetic signal as they are also present in taxa situated in different clades of the miltogrammine phylogenetic tree, and they are symplesiomorphic at this level 1,2,9,12 . Additionally, the shape of the oral ridges and the general morphology of the anal division of Ae.   12 . Unfortunately, the first instar larva of M. nudibasis, the sister species to Aenigmetopia in the phylogenetic tree provided by Johnston et al. 12 , remains unknown. Therefore, the possible paraphyly of Metopia cannot be tested by larval morphology, and formal synonymization of Aenigmetopia under Metopia is considered premature and should await further analyses based on additional taxa and more data. The first instar of Am. auriceps is almost identical to those of the Palaearctic species in most characters 2 , including: (1) long and slender first sensillum basiconicum situated close to the central cluster of sensilla, (2) ventral organ sensilla placed on a large fleshy lobe, (3) oral ridges short, (4) labrum long, curved downward, gradually tapering to pointed anterior tip, (5) broad basal part of mouthhook, (6) cuticular sculpture on abdominal segments in the form of warts, (7) abdominal segments ventrally with locomotory protuberances/prolegs, (8) abdominal segments dorsally with transverse row of large triangular cones, (9) peristigmatic tufts small, and (10) spiracular field without encircling spines. The first instar of Am. auriceps differs from the majority of Palaearctic Amobia in two key characters, which henceforth should be considered to be intragenerically variable: (1) the antennal basal ring of Am. auriceps more pronounced than the low and flat structure described in the larvae of Palaearctic species; and (2) the entire lateral surface of thoracic segments in Am. auriceps covered by cuticular warts (Fig. 2B), whereas at least in the Palaearctic Amobia the lateral surface of the first thoracic segment is covered by dense cuticular ridges 2 .
The first instar larva of species representing the genera Eremasiomyia, Protomiltogramma and Pterella share the following set of character states 2,3 : (1) basal ring in antennal complex absent, (2) first sensillum basiconicum shifted to the inner periphery of maxillary palpus, and (3) posterior surface of anal division between spiracular field and anal complex with circular depression. This set of features is not observed in larvae of other miltogrammines, and Eremasiomyia, Protomiltogramma and Pterella probably form a monophylum. The morphology of the first instar larvae of Australian Protomiltogramma is almost identical to that of the Palaearctic species P. fasciata (Meigen) in all aspects, such as the form and distribution of the sensory organs on the pseudocephalon, the cephaloskeleton, the cuticular sculpture and the shape of the anal division. The presence of a large circular depression on the pseudocephalon and on the anal division represent two intriguing character states of Protomiltogramma larvae. Szpila 2 did not mention these depressions in his description of the larva of P. fasciata, but they are plainly visible in his Fig. 56, and they have also been observed in larval material of Protomiltogramma from the United Arab Emirates (K. Szpila, unpubl.). These depressions have been described only in the genera Protomiltogramma (three species, depressions on both pseudocephalon and anal division), Pterella Robineau-Desvoidy (two species, depression only on anal division) and Phrosinella Robineau-Desvoidy (three species, depression only on anal division) 1,2,17 . They are of a very similar morphology and therefore unlikely to be artefacts. However, the function of these circular depressions remains unknown and needs further study using other approaches, e.g. anatomical methods.
The present study fills an important gap in our knowledge both of the preimaginal instars of miltogrammine flies and the morphological diversity of endemic Australian taxa. Congruence was established between larval morphology and the current phylogenetic hypotheses presented for the Australian Miltogramminae 9,12 and the global Miltogramminae 1,18 . This alignment between larval morphology and molecular phylogeny in the Australian Miltogramminae provides evidence for both the stability of these traits in the Sarcophagidae and their potential value for the inference of evolutionary relationships in this family.

Methods
Adult female miltogrammine flies were collected from various locations in the Northern Territory, Australia (details available in the "Results"). Once preliminarily identified, freshly caught females were killed with ethyl acetate vapour and transferred to a stereomicroscope. Live first instar larvae were then obtained by gently squeezing the female's abdomen. Following larval extraction, females were either pinned (Protomiltogramma) or preserved in 95% ethanol (Aenigmetopia, Amobia). Females were identified using the keys of Malloch 19 , Johnston 8 and Johnston et al. 9,11 in combination with comparative studies of voucher specimens from the Australian National Insect Collection, Canberra, Australia (ANIC); the Australian Museum, Sydney, Australia; the Department of Ecology and Biogeography, Nicolaus Copernicus University, Toruń, Poland (DEB); and the Natural History Museum of Denmark, University of Copenhagen, Denmark.
Larvae were killed by soaking in hot water (about 95 °C) to avoid deformation and stored in 70% ethanol. Preserved larvae were slide-mounted in Hoyer's medium for light microscopy. Preparation for scanning electron microscopy (SEM) involved dehydration through 80, 90 and 99.5% ethanol and critical-point drying in CO 2 , after which the larvae were coated with ~ 100 nm of platinum/palladium. SEM images were taken with the use of a JEOL JSM 6335F field emission microscope. Light microscope illustrations were produced from photographs made with the use of a digital Nikon 8400 camera mounted on a Nikon Eclipse E200 microscope.
Material intended for visualization with confocal laser scanning microscopy (CLSM) was freshly prepared from whole available larvae. Specimens were cleared initially by immersion in 10% potassium hydroxide for over 48 h, until tissue digestion was satisfactorily obtained. The material was then dehydrated in 99.5% EtOH Scientific Reports | (2021) 11:2687 | https://doi.org/10.1038/s41598-020-80139-x www.nature.com/scientificreports/ for 20 min and the specimens were embedded in Euparal on a microscope slide and covered with a coverslip.
Due to the small size of the examined material, flat microscope slides were used to avoid damaging the structures of interest. Prepared slides were scanned using a Leica TCS SP8 Confocal Laser Scanning Microscope. In order to obtain optimal fluorescence, only the excitation wavelengths of 561 nm and 633 nm were used. A strong fluorescence signal was also obtained with an excitation wavelength of 488 nm, but this was omitted as it simultaneously revealed some contamination and undigested tissue, which significantly disturbed the resulting image. First instars of Ae. amissa and Am. auriceps were similar in size and examined under a 63× objective lens with a numerical aperture (NA) of 1.4, and the first instar of P. cincta was examined under 40× with a NA of 1.3. Effective 3D visualization requires collecting more data than necessary to obtain acceptable 2D images 20 , and differences in the thickness of the individual larvae required optimization of the amount of data collected from each frame. Individual z-step size settings were defined for each sample manually. The larger size in the z-plane imposed the collection of fluorescence signals from different focal planes, which allowed for full visualization of the examined structures. After the acquisition of the preliminary image series, the 3D model was built in the LAS X 3D Viewer program. When the received model was of low quality acquisition of image series was repeated with manually increased number of z-steps. The respective number of z-frames was individually adjusted for each of the three specimens; for Ae. amissa there were 123 z-frames, for Am. auriceps 146 z-frames and for P. cincta 224 z-frames. The females of P. cincta and P. plebeia, all larvae, and associated microscope slides have been deposited in DEB. Females of Ae. amissa and Am. auriceps have been deposited in ANIC. Larval terminology follows Courtney et al. 21 , with a few modifications proposed by Szpila and Pape 22 and Szpila 2 . Nomenclature follows Pape 6 and Johnston et al. 9,11 , with the generic names of Aenigmetopia and Amobia abbreviated as Am. and Ae., respectively, to avoid confusion. Label data of specimens are given verbatim using a slash (/) for the start and end of a line, and a double slash (//) for the end of a label and the beginning of the next (from top to bottom on the same pin) (Fig. 6).