Development of the indirect flight muscles of Aedes aegypti, a main arbovirus vector

Figures

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  Fig. 1. IFM precursors in A. aegypti larvae, DVM. a–c Coronal sections of late L3 thorax hematoxylin and eosin stained showing three myoblast nests transversely cut. a, b Those three myoblast clumps per hemithorax were labeled as 1, 2 and 3, which correspond to future formation of DVM I, DVM II and DVM III, respectively. Adepithelial cells (ae) next to the basal side of the imaginal wing disc epithelium (ed), are observed. c, d Myoblast morphology during L3 instar. Inset (d) showing diversity of morphology among such as “teardrop”, spindle shapes and dividing cells (arrows). e, f In early fourth-instar larvae developing DVM, initiate de novo formation by fusion of FCM to FC establishing multinucleated areas (yellow asterisk in e) to form primordial myotubes surrounded by FCM. Fascicles 1, 2 and 3 show four, five and four (or five) fusion sites, respectively, showing large nuclei in the center of each primordial myotubes. f The myotubes continue their development to form multinucleate myotubes, where fascicles 2 and 3 have 5 and 4 myotubes, respectively, with abundant myoblasts among them. g, h During late fourth-instar larvae the myotube formation is almost complete, where most…

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  Fig. 2. Dorso-coronal sections of the thoracic region of late L4 show fascicles corresponding to DLMs. Myotubes in each fascicle are showed in oblique sections. a, b Each DLM fascicle (three each side, right and left, labeled as 1, 2 and 3) is formed by four myotubes. a At this section level fascicles 1 are attached by the anterior side to the thoracic epithelium in upper thorax (black arrowheads). b In this plane attachment of DLM fascicles numbered 2 and 3 to the thoracic epithelium is to the lower thorax (black arrowheads) and fascicles 1 are out of focus in the histological section. c Detail of attachment region of DLM myotubes 3 and 2, where myotendinous junctions (MTJ) are observed (black arrowhead; d and d′, dorsal). d Detail of fascicle 3 where possible fusion competent myoblasts (fcm) are observed between four myotubes. e DLM primordia in the thoracic region of L4 instar. At this oblique section the three fibers per hemithorax are captured (numbered 1, 2, 3), each fascicle formed by four fibers (red asterisks). Those fascicles corresponding to the LOMs or scaffold myotubes, possibly in splitting process. Several myoblasts among fibers are observed (spots among fibers).…

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  Fig. 3. Actin assembly and organization in multinucleated primordial IFMs. a–d IFM primordia of early and late phases from L3 and L4 instars were stained with Rhodamine-Phalloidin (red) to highlight actin filaments and nuclei with DAPI to visualize nuclei (blue). a In early L3 primordium, disperse Phalloidin-labelled short actin filaments widely distributed were observed. b In late L3 instar, the muscle primordia showed abundant actin. At this stage primordia showed stripes forming sections-like along the primordium (dotted lines). c The early L4 primordium, actin polymerization led to the formation of immature myofibrils organized in bundles within myotubes. d The late L4 primordium showed a larger size and abundant immature myofibrils forming muscle fibers. At this stage, the nuclei were observed forming rows along the muscle fibers and, some of them showed a larger size than the others (dotted circles). Insets show enlargement of dashed boxes. Scale bar: 100 μm

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  Fig. 4. F-actin and myosin filaments patterns in DVM primordia. L4 DVM primordia obtained from L4 instar was stained with TRITC-Phalloidin (red), DAPI (blue), and anti-Myosin Heavy Chain antibody (MHC) (green) to observe the F-actin and nuclei distribution in forming myofilaments. Images of optical slices were collected by confocal microscopy and a projection constructed using the ZEN program (Zeiss). a A structure showing hundreds of immature myofibrils forming five possible muscle fibers (1–5, clearly separated). The DVM primordium showed an approximate size of 300 μm in both length and width. b Nuclei forming rows within the primordium. c The same primordium showing hundreds immature myofibrils formed by F-actin whitin muscle fibers. Also, both ends of primordium were intensely stained with actin. d Myosin distribution in primordium shows distribution parallel to actin. d Merge. Scale bars: b–e 100 μm

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  Fig. 5. Myoblast fusion in IFM development. L4 primordia were dissected and analyzed by TEM to show details of cell structure and organization during IFM developing. a, b During early fusion steps prefusion vesicles are formed and coalesce (red arrowheads) close to the membranes apposition zone. b Magnification of white dotted box in a, where small vesicles were observed; inset, magnification of dotted box showing vesicles surrounded by electron-dense material. c, d A rounded myoblast showing membrane extensions such as filopodia-like (green arrows), another cell shows a podosome-like protrusion (blue arrowhead) and, at cell contact sites some fusion pore-like structures were also observed (red arrowheads). d Magnification of white dotted box shows detail of the filopodia-like structures (green arrows), and fusion pores, where some vesicles between the pores-like were seen (light areas between red arrowheads). e, f Fusing myoblasts membrane apposition zone where possible pores are forming and the cytoplasm is crossing from one cell to another are shown (red arrowheads). f Magnification of white dotted box in e, showing possible cytoplasmic continuity among two cells through two pore-like…

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  Fig. 6. Actin organization during developing IFMs in L3 and L4 stages. Myofibrils formation during IFM development was analyzed by TEM. a In late L3 primordia, short actin filaments arrays (arrows) were observed. b Early L4 primordium showed long filaments. c Late L4 primordium showed greater number of long and dense fibers, possibly immature myofibrils. Scale bar: 2 µm

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  Fig. 7. Myofibrils and sarcomere maturation during pupae and adult stage. The maturation process of myofibrils and sarcomeres was analyzed by confocal microscopy, staining F-actin with rhodamine-phalloidin (red) and nuclei with DAPI (blue). a In late pupal stage, the myofibrils had a syncytial structure, with nuclei showing a relaxed chromatin organization (white arrows). b In adults, uniform mature myofibrils containing long and organized mature sarcomeres were found. In both pupae and adult IFMs, the nuclei forming rows were observed. c The isolated myofibrils showing the classical structure of mature sarcomere, including the bright Z disc and the dark M-line (magenta and yellow arrowheads). d Magnified image of sarcomere structure. Scale bars: a–c 50 µm; d 10 µm

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  Fig. 8. Sarcomere ultrastructure in pupae and adult IFMs. IFMs from A. aegypti pupae and adult were dissected and analyzed by TEM. a In longitudinal sections of pupae muscle, still in growth, short sarcomeres well-structured (~ 1.95 μm), showing the Z-discs and M-lines (magenta and yellow colors, respectively) were observed. Large and elongated mitochondria were seen. b Adult mosquito muscles showed sarcomeres completely formed (~ 2.90 μm), with clearly defined Z-discs and M-lines. Numerous giant mitochondria with tightly compressed cristae were found alongside the flight muscles. c Cross section of a mature myofibril showing the sarcomeres with hexagonal arrangement. d Magnified image of the dashed box in c, showing thick myofilaments (myosin, visualized as large hollow circles), each surrounded by six thin myofilaments (actin, visualized as small black spots surrounding each thick filament), in a relation of 6:1. Scale bars: a 1 µm; b 2 µm; c 0.5 µm