Achanthiptera rohrelliformis (Robineau-Desvoidy) (Figs 1A, B, 2A–F, 3A–G, 4A–I): 2 third-instar larvae, labelled: “C.27:1”; BMNH — 15 third-instar larvae, labelled: “April 1912 / Kryger / W. Lundbeck col.”; NHMD — 35 second- and 27 third-instar larvae, labelled: “Berlin / Jungfernheide / 1909”; ZMB — 8 third-instar larvae, labelled: “Berlin / Wittenau / 1911”; ZMB

Potamia littoralis Robineau-Desvoidy (Figs 1C, D, 5A–C, E, G, 6A–I): 5 second- and 8 third-instar larvae, labelled: 21 Jul. 2013, Pławin, Poland, A. Grzywacz leg.; NCUT

Australophyra rostrata (Robineau-Desvoidy) (Figs 1E, 5D, F, H, 7A–I): 5 third-instar larvae, labelled: 1 Feb. 1995, Kuitpo Forest, South Australia, Australia, J. F. Wallman leg.; NCUT

To avoid repetition, general morphology of all species is described jointly, and the species-specific details are highlighted in the following subsections. Due to the poor condition of the second-instar larva of Ac. rohrelliformis, it was only feasible to examine its cephaloskeleton using CLSM.

Pseudocephalon. Bilobate and equipped with paired antennal complex (an), maxillary palpus (mp) and ventral organ (vo) (Figs 3B, 6B, 7B). Functional mouth opening surrounded by facial mask consisting of numerous oral ridges (or) and a pair of labial lobes (ll) equipped apically with sensilla of the labial organ (lo) (Figs 3B, E, 6B, 7B). Anterior margin of the facial mask with a row of cirri (cir). Antennal complex with conical antennal dome (and) situated on antennal basal ring (abr) (Figs 3C, 6B, 7B); the length of and half the height of abr (Figs 3C, 6B, 7B). The mp consists of three sensilla coeloconica (sc), three sensilla basiconica (sb), several small additional sensilla arranged in a tight cluster and two sensilla coeloconica of non-maxillary origin (ns) positioned laterodorsally (Figs 3D, 6C, 7C). The bulge-shaped vo equipped with four sensilla (Figs 3B, E, F, 6D, 7D) and placed at the anterior margin of or (Figs 3B, E, 6B, 7B).

Cephaloskeleton. Paired mouthhooks (mh), unpaired intermediate sclerite (is) and basal sclerite (bs) (Figs 1A–E).

Second instar with several well-sclerotized, ventrally directed suprabuccal teeth (sub) (Figs 2A, 5A) placed anterior to mh. The mh L-shaped in lateral view (Figs 1A, C, 2A, 5A) with slender base and distal parts symmetrical and closely appressed (Fig. 2B). Apical part of mh enveloped in the dome-shaped anterior rod (aro) (Figs 1A, C, 2A, B, 5A). Dental sclerite (ds) and irregular accessory stomal sclerites (acc) located below each mh (Figs 2A, 5A). Convex epistomal sclerite (es) and a pair of labial sclerites (ls) placed anteriorly between anterior arms of is (Figs 2A, 5A, B). Paired rod-like rami (r) lie freely between lateral arms of is (Figs 2B, 5B). Basal sclerite (bs) with paired vertical plates (vp), with dorsal (dc) and ventral cornua (vc) connected anteroventrally by strongly sclerotized ventral bridge (vb) (Figs 1A, C, 5A, B) and anterodorsally by tightly appressed horseshoe-shaped dorsal bridge (db) (Figs 1A, C, 5A). The vp broad, dc shorter than vc (Fig. 1A, C). Posterior parts of dc and vc poorly sclerotized, the latter with dorsal extension (de). Hypopharynx with distinctly developed longitudinal ridges.

Third instar with asymmetric, closely appressed mh, the left being shorter (Figs 2C, D, 5C, D). Distal parts curved ventrally (Figs 1B, D, E, 2C, D, 5C, D). Triangular ds and acc situated ventrally to base of each mh (Figs 2C, D, 5C, D). The ds connected with base of mh by slender, sclerotized hinge along anterodorsal margin (Figs 2C, D, 5C, D). Elongated and convex es and paired ls lie freely between anterior arms of is (Figs 2E, F, 5E, F). The is H-shaped in dorsal view (Figs 2F, 5E, F). Parastomal bar (pb) fused with dorsal margin of is, creating anterodorsal extension (Figs 2E, 5F, G, H). Paired well-sclerotized, rod-like rami (r) lie between arms of is (Figs 2F, 5E, F). The bs consists of paired, broad vp each with dc and vc (Fig. 1B, D, E). The vp connected anteroventrally by vb and anterodorsally by db (Figs 1B, D, E, 2F, 5E, F). Hypopharynx with longitudinal ridges.

Thoracic and abdominal segments. Anterior spinose band (asb) on first thoracic segment (t1) broad and complete (Figs 3A, B, 6A, B, 7A, B). Spines of asb colourless and arranged densely in long rows, closely adjacent ventrally and slightly separated dorsally (Figs 3A, 6A, 7A). Anterior third of t1 with transverse cleft (cl), followed ventrally by several rows of spines (Figs 3A, 6A, 7A). Each thoracic segment ventrally with a pair of trichoid sensilla of Keilin’s organ (ko) (Figs 3B, 4B, 7A, E). Second (t2) and third (t3) thoracic and first abdominal (a1) segments in second instar with single short row of fine spines. Third instar with complete anterior spinose bands on t2 and t3 arranged in relatively short rows (Figs 3A, 6A, 7A). Abdominal segments (a2–a7) anteroventrally with creeping welts (vcw) and anal division (ad) with pre-anal welt (pre) (Figs 4C, I, 6F, I, 7F, I). Elliptical lateral creeping welts (lcw) present between abdominal segments, barely visible, never covered by spines (Fig. 7F). The posterolateral margin of a1 with circular bubble membrane (bm) of many globules clustered together and level with the adjacent integument (Figs 4D, 6F, 7F). All spines of asb on t1–t3 and a1–a3, as well as of vcw on a2–a7 directed posteriorly (Figs 3A, B, G, 4A, C, 6A, B, F, 7A, B, F), while spines of pre point anteriorly (Figs 4I, 6I, 7I). Scars from muscle attachments strongly visible on surface of all thoracic and abdominal segments.

Anal division. Ventrally with anal opening (ao), porous anal plate (ap) and distinct anal papillae (Figs 4I, 6I, 7I). An unpaired post-anal papilla (pa) present directly posterior to ao. Paired sub-anal (sa), para-anal (paa) and extra-anal (ex) papillae shifted anteriorly relative to the pa (Figs 4I, 6I, 7I). Each sa with a sensillum basiconicum and two sensilla resembling sensilla ampullacea. Spiracular field with seven pairs of papillae (p1–p7) and a pair of posterior spiracles (ps) located centrally (Figs 4G, H, 6H, 7H). Each papilla composed of a sensillum plus the surrounding area. Papillae p1, p3, p5 and p7 with a sensillum basiconicum. Papillae p2, p4 and p6 indistinct, placed ventral to the spiracular field and equipped with a sensillum ampullaceum.

Phylogenetic implications based on larval morphology have been discussed for Muscidae by several authors (Roback 1951; Schumann 1954; Ferrar 1979; Skidmore 1985), and, among all larval structures, the cephaloskeleton has been identified as of particular importance. For instance, having observed a striking resemblance of the cephaloskeleton, including the shape and arrangement of sclerites between the larvae of P. littoralis and Hydrotaea dentipes (Fabricius), Hennig (1965) ultimately concluded that Potamia was more closely related to Hydrotaea than to Phaonia Robineau-Desvoidy. The asymmetry of the mouthhooks in the third-instar larva supports the placement of Achanthiptera as well as Potamia and Australophyra within the Azeliinae + Muscinae clade (Grzywacz et al. 2021). All species examined in this study are nested within the subfamily Azeliinae and have larvae closely resembling those azeliines for which descriptions and drawings are available. Similarities in general morphology and in the details of the cephaloskeleton and anal division with the dentipes-group within Hydrotaea are particularly compelling (Skidmore 1985; Grzywacz et al. 2014). Shared features are the shape of the anal division: angular outline of the segment in lateral view, spiracular field directed posterodorsally often with dorsal-most part of ad distinctly above the level of the dorsal surface of preceding abdominal segments, a W-shaped anal plate with all anal papillae well developed, mostly bulge-like, as well as posterior spiracles with straight to sinuate respiratory slits in a parallel to radiate arrangement (Skidmore 1985; Grzywacz 2013b; Grzywacz et al. 2014). In Muscinae, the spiracular field is directed posteriorly and the dorsal surface of the segment is not above the level of preceding abdominal segments, the anal plate is either small to enlarged, with partially reduced anal papillae, while the slits of the posterior spiracles are serpentine to torturous and arranged in a peripheral to encircling configuration (Skidmore 1985; Grzywacz 2013a). As for the differences in the cephaloskeleton, Azeliinae larvae usually possess well-developed accessory oral sclerites, while in larvae of the Muscinae these are absent or reduced (Skidmore 1985; Grzywacz 2013b; Grzywacz et al. 2014). In addition, the intermediate sclerite of Azeliinae is equipped with a visible anterodorsal extension derived from the reduced parastomal bar, while in Muscinae this sclerite is short and robust, and its dorsal surface is strongly modified (Fig. 8E, F).

Our results provide the first description of the morphology of the second-instar larva of Ac. rohrelliformis and P. littoralis. The species exhibit numerous similarities in their cephaloskeletons. Notably, the presence of a dome-shaped anterior rod closely attached to each mouthhook differentiates them from other muscid species. CLSM images revealed that each rod envelops the tips of the mouthhooks and further extends forward in relation to them. To the best of our knowledge, this specific shape of the anterior rod has not been documented previously and its function remains uncertain. Although previous studies have provided relatively detailed descriptions of spinulation pattern, arrangement of anal papillae and shape of posterior spiracles in the third-instar larva of Ac. rohrelliformis, P. littoralis and Au. rostrata, details of the cephaloskeleton have either been briefly described or not described at all. Illustrations of the third-instar larval body, the cephaloskeleton and the posterior spiracles were provided by Lobanov (1975) for Ac. rohrelliformis, by Zimin (1948) for P. littoralis, by Fuller (1932) and Zumpt (1965) for Au. rostrata and by Skidmore (1985) for all three species. Examination of these figures reveals that the authors observed all sclerites recognised in this study, except for the epistomal sclerite, parastomal bars, a pair of labial sclerites and a pair of rami in all examined species as well as the rectangular accessory process in Ac. rohrelliformis. Additionally, CLSM revealed that the dental sclerite in all examined species is connected to the base of the mouthhook by a narrow, sclerotized hinge. Skidmore (1985) noted that the intermediate sclerite of Ac. rohrelliformis, P. littoralis and Au. rostrata is equipped with a “very strong acute dorsal tooth”, which corresponds to the location of the parastomal bar that has fused with the dorsal edge of the intermediate sclerite (Walczak et al. 2022). Despite the long-standing belief that the absence of a parastomal bar was considered a distinctive feature of the family Muscidae (Schumann 1954; Ferrar 1979; Skidmore 1985), recent evidence has demonstrated its presence in reduced form in muscid species (Walczak et al. 2022, 2023). Most importantly, all authors correctly observed the presence of additional sclerites below the apical part of the mouthhooks, however they did not recognise the asymmetry of the oral bars and anterior rods in Ac. rohrelliformis, as well as the peculiar shape of anterior rods in both Ac. rohrelliformis and P. littoralis. The right mouthhook in Ac. rohrelliformis is fused basally to a serrated oral bar through a rectangular accessory process, while its apical part is closely attached to a spade-like anterior rod. Below the left mouthhook, these structures are reduced, irregular and unconnected to the base of the mouthhook. Although asymmetry of the mouthhooks is a feature shared by all species in the subfamily Azeliinae (Grzywacz et al. 2021), the pronounced asymmetry of the accessory oral sclerites documented in this study has not been reported previously. While the mouthhooks and accessory oral sclerites do not exhibit such pronounced asymmetry in P. littoralis, they do possess comparably well-developed accessory oral sclerites as observed in Ac. rohrelliformis. This is in comparison with Skidmore (1985), who misinterpreted the anterior rod and oral bar in P. littoralis as “very slender, short and weak”. A similar inconsistency exists in the description of the morphology of P. scabra (Giglio-Tos). Skidmore (1985) mentioned the absence of accessory oral sclerites in this species, misinterpreting the line drawings presented by Calhoun et al. (1956), and thus considering P. scabra a sole representative of Azeliinae devoid of accessory oral sclerites. A re-examination of fig. 3 in Calhoun et al. (1956) revealed that the third instar of P. scabra has accessory oral sclerites that are as well-developed or even more strongly developed than in P. littoralis. Similarly to Ac. rohrelliformis, the oral bar of P. scabra closely adjoins the basal part, and the anterior rod adjoins the apical part of the mouthhook. All aforementioned morphological structures, in particular the shape of accessory oral sclerites, and the connection between them, might have been overlooked by previous authors due to the limitations of light microscopy techniques. On the other hand, this might also have resulted from the process of microscopical preparation itself. This study revealed how maceration time in potassium hydroxide may critically impact the visibility of fine sclerites, making them nearly invisible. Individually adjusted maceration time is therefore strongly recommended for each specimen under study. Furthermore, Skidmore’s cephaloskeletons of Ac. rohrelliformis and P. littoralis were dissected from puparia, which could have caused the displacement or rupture of small, weakly sclerotized morphological structures.

The function of the modified anterior rods in Ac. rohrelliformis and P. littoralis remains ambiguous, and further investigation of larval biology would be necessary to unravel its significance. Keilin (1917) demonstrated a close relationship between larval feeding habits and the presence/absence and shape of cephaloskeletal elements, which was later refined by other authors (Keilin and Tate 1930; Thomson 1937; Roberts 1971; Ferrar 1979; Skidmore 1985). Based on the presence of additional oral sclerites and following the conclusions of previous authors, larvae of Achanthiptera, Australophyra and Potamia should be considered facultative carnivores. This larval feeding strategy is particularly indicated by relatively blunt-tipped mouthhooks, well-developed accessory oral sclerites composed of oral bars and anterior rods and well visible longitudinal ventral pharyngeal ridges. Accessory oral sclerites are known to play a vital role in facilitating the movement of mouthhooks when piercing the cuticle of the prey (Roberts 1971). When the mouthhooks are flexed downwards, the oral bars emerge from the functional mouth opening being held in place by the anterior rods to grip the cuticle of potential prey, as shown in fig. 5H in Grzywacz et al. (2014) (Roberts 1971; Skidmore 1973). While accessory oral sclerites in Au. rostrata resemble those of the majority of species within the genus Hydrotaea (Grzywacz et al. 2014), their modified shape in Achanthiptera and Potamia seems unlikely to support this function. It is therefore proposed here that the modified anterior rods in the second- and third-instar larvae of both Achanthiptera and Potamia, in conjunction with the mouthhooks, increase the surface area for collecting food mass. A similar modification for increasing the surface area of the mouthhooks for shovelling liquefied food has already been reported in some saprophagous and coprophagous species within the subfamily Muscinae. For example, species of Musca Linnaeus, Neomyia Walker and Stomoxys Geoffroy are known to have a laterally expanded apical part of the mouthhook (Fig. 8A–D), enabling more efficient collection of food mass (Ferrar 1979; Skidmore 1985). In Atherigona Rondani, in turn, a distinct modification of accessory oral sclerites is well known, reflecting the adaptive transition from facultative carnivory (e.g. At. culicivora Kovac, Pont and Deeming and At. orientalis Schiner) to phytophagy (e.g. At. reversura Villeneuve) (Skidmore 1985; Grzywacz and Pape 2013, 2014; Kovac et al. 2023). In the subgenus Atherigona s. str., the massively enlarged oral bar and the reduced mouthhook are fused, thereby functioning as a single structure. As a result, lateral movement is inhibited, and the fused sclerites functionally resemble the broadened mouthhooks of phytophagous Anthomyiidae (Roberts 1971; Ferrar 1987).

In summary, based on the current study and literature data, the larval instars of Au. rostrata are considered facultative carnivores, while strong asymmetry of mouthhooks and modified accessory oral sclerites in Ac. rohrelliformis indicate a saprophagous lifestyle. The larval morphology of the second and third instars of P. littoralis closely corresponds to that of Ac. rohrelliformis, the only difference being that in P. littoralis both oral bars lie freely. Nonetheless, considering similar modifications of accessory oral sclerites, we assume that both P. littoralis and P. scabra are saprophages, although this requires corroboration, such as through observations from rearing experiments.