For mosquitoes, flying is fundamental for survival, population dispersion and disease transmission, and there are vector control strategies based on interfering with mosquito muscle formation with significant effects on disease transmission [47,48,49]. Notwithstanding, despite the importance of mosquitoes as vectors of infectious diseases, which are major health concerns, the knowledge of the development of the IFMs, that give the potency for wing beating [50, 51] is limited. In contrast, in D. melanogaster there are numerous morphologic and molecular biology studies about IFM development, constituting a wide study field with numerous tools to study it, including mutants, antibodies and manipulation methods [45]; these tools are necessary to develop for mosquito studies but the knowledge about fly muscle development was very helpful for our study.

In this work we present an initial description of A. aegypti IFM development. It is important to mention that we found notorious differences in the development of A. aegypti, belonging to suborder Nematoceran (a “lower” dipteran), with those of D. melanogaster, a Brachyceran (a “higher” dipteran), and we found interesting similarities to Chironomus sp., which is another lower dipteran Nematoceran, including the timing of the muscle development [52,53,54]. In D. melanogaster, as in all higher dipterans, there are three larval stages, followed by a puparation, with a prepupal stage and, after, during the metamorphosis (pupation) most larval muscles are dissolved, and formation of adult muscles, including IFMs is accomplished. In comparison, in A. aegypti and Chironomus sp., there are four larval stages and the IFM development begins actively during the last two and proceed during pupation. We were able to identify in A. aegypti, as early as L3, clusters of cells that will form the IFMs.

In dipterans there are two IFM groups of fibrillar muscles, with major differences during development: the DLM constructed priming on larval oblique muscles (LOMs), which persist after metamorphosis and are used as templates for myoblasts migration and fusion to form the adult muscle; and DVM, formed de novo by migration of myoblasts to specific locations where they conglomerate around founder cells [55, 56]. Here, we show that at the front of L4 A. aegypti thorax, DLM are formed of three fascicles per hemithorax, attached to frontal thorax at different levels, with a structure similar to LOMs to which myoblast-like migrate and fuse, then these muscles possibly split to originate six myotubes per hemithorax, similarly to those happening in D. melanogaster and Chironomus sp. [20, 45, 52,53,54,55] and, this fascicle conformation coincides with images presented before in A. aegypti (cf. [46]). In comparison, the DVMs are constructed de novo, and our images strongly suggest the migration of FC and FCM, which have been proposed that originate from wing imaginal disk cells [20], forming primordia [52, 53], which will evolve to form myotubes, myofibers and sarcomeres. Interestingly, we observed that the putative IFM primordia of A. aegypti, where primordial myotubes are contained, were enveloped with a basement membrane, as has been observed in Chironomus sp. [52, 53]. When the IFM precursors and adult muscles are compared between D. melanogaster and A. aegypti, major differences are found, including the structure of primordia and the final number of fibers in the adult (Additional file 1: Fig. S1 and Table 1) [10, 20, 57,58,59,60,61]. In a previous report, the number of adult IFM fibers in A. aegypti, was compared among a population of an inbreed laboratory strain with three field collected populations, and significant differences in the number was observed, and differences were more frequent in DVMs of laboratory strains. Furthermore, the authors detected DLM splitting of muscles leading to side number asymmetry and formation of ramified adult muscles, which probably affects the ability to fly. The higher variation in laboratory strains is explained by arguing that in nature the modified individuals are eliminated in a “stabilizing selection” [10]. These results coincide with our observations where the number of DLM fascicles was constant, but in DVMs in the groups 1 and 2, and despite the small number of samples analyzed, variations in myotube numbers were seen [56]. The regulation of muscle size and number during IFMs formation in D. melanogaster is controlled by a balance between fusion and proliferation during larval and pupal stages [56, 62,63,64].

In this work, in A. aegypti larvae we recognized three groups of cells (primordia) at DVM locations as early as L3, which we followed through L4 and pupa up to generate the adult muscles by hypertrophic growing. Analyzing the DVM development in A. aegypti we observed cells with locations and “teardrop” and spindle morphologies, corresponding to D. melanogaster FCM which migrate from the wing imaginal disc to specific sites where primordia will be developed forming nascent fibers. Our observations of FCM-like cells in A. aegypti DVM primordia allow us to reasonably propose, that in this mosquito FCM cells go through a similar process with active division, migration and location out and inside the primordial myotubes [62]. In addition, in late L3 and early L4 the FCM associate to founder cells, which have distinguishable big and heterochromatic nuclei at the center of nascent myotubes, and, as result of cell fusion, syncytial myotubes are formed.

Another kind of putative primordium was observed in the expected location, in respect D. melanogaster and Chironomus sp., for the TDT muscle and, as it was expected, it has a different organization, without myotube divisions, corresponding to a tubular muscle. Tubular muscles have major differences in respect to DVM, as we observed here, including the radial distribution of nuclei and FCM fused in a single syncytial compartment and a variable number of fibrils (Table 1) [65,66,67].

In D. melanogaster, it has been reported that mechanical tension during the muscle formation depend on an attachment process, via myotendinous junction (MTJ) formation, which is necessary to stabilizing the myofibrils during assembly, and become innervated by motoneurons through neuromuscular junctions to mature into stable and functional muscles [63, 68,69,70]. In A. aegypti we observed at L4 three DLM fascicles per hemithorax bound to the anterior epithelial wall of the thorax. At this stage, siphons are visible, DLM are attached to the epithelium only at the front of the thorax, close to the respiratory trumpets by mean of elongated extensions of tendon cells. In this study we did not observed the fixation of DLM to the posterior thorax lamina, which in D. melanogaster is formed by tendon attachments that are early stablished at both muscle sides, maintaining tension during development [68,69,70]. In addition, referring to the mechanism of MTJ formation in A. aegypti, we observed that at posterior DLM end, there are cells with enlarged nuclei, like those that contact with thoracic epithelial tendon cells at anterior end, besides possible MTJ in formation (Fig. 2e, green arrowheads). Furthermore, at L4 primordia of both DLM and DVM, were observed actin and tubulin rich plates at both ends of muscle fibers (data non shown), which could be related to the construction of MTJ, as it has been reported for D. melanogaster [68,69,70]. Many issues are open in A. aegypti in respect to MTJ and neuromuscular junctions in formation as the identification of integrins and other extracellular matrix molecules participation [39, 63,64,65,66, 68,69,70,71,72,73].

At L4, the three DLM have four myotubes and this number was constant and is in accordance with images previously reported (Fig. 2 and cf. [46]). In contrast, in DVM we observed that the numbers of myotubes for DVM 1, 2 and 3 are majorly 4, 5 and 4, respectively, but some variation was observed, in agreement with previous authors and it is difficult to explain [10].

Interestingly, DLM location in L4 coincide with the site of expression of reporter genes under the control of Aeact-4 promoter, a female-specific promoter used for transgenic mosquito construction of flightless phenotype in females of Aedes spp. for vector control [48, 49], suggesting that this promoter rules expression specifically in the DLM. In addition, the expression ruled by Act88F from D. melanogaster, when cloned in the Culex quinquefasciatus mosquito, directed the expression of a reporter in all the IFMs [74], and the D. melanogaster promoter for Act79B gene directs the expression specifically in TDT in the fly [65], indicating the diversity and specificity of the promoters involved in IFM development in dipterans, demonstrating that there are general and specific promoters for IFM and other thoracic muscles and, research of this issue will be important for designing new constructions aimed to vector control.

During A. aegypti L3 instar, myoblasts migrate to form small clusters located at defined places, priming the construction of DVM. Inside the nascent fascicles, founder cells (FC) define the myotube formation recruiting fusion competent cells (FCM), which divide actively, and it is possible to recognize their presence by their characteristic morphologies, that are considered hallmarks of this kind of fusing cells [62]. Myoblast fusion process involve the formation of filopodia and podosome for the initial contact and the organization of prefusion vesicles that coalesce around points where the integration of myoblasts to the myotubes are happening [30, 32,33,34, 74], generating syncytial structures. Here we present in A. aegypti images similar to those of fusion events during D. melanogaster myotubes formation and concomitant to this, prefusion vesicles which could conduct to the formation of syncytial-fused tubes with aligned nuclei forming rows in the muscle axis during early formation of contractile adult muscle [32,33,34, 54, 62, 75].

For the organization of muscle components during development actin, myosin and tubulin are fundamental for sarcomere construction [39,40,41,42, 76, 77]. General organization of F-actin and myosin was studied during the A. aegypti IFM myogenesis. F-actin abundance increase from L3 to pupa and organization evolved from short structures in early L3 muscle primordia, in the cells that are not yet fused. After, in late L3, when the cells fusion is in progress, F-actin was structured in short filaments, and at L4 F-actin fibers are longer and along the myotubes. Simultaneously, in L4, muscular myosin was strongly produced and associated to actin filaments, forming premyofibrils. Furthermore, in A. aegypti IFM L4 primordia we observed dense longitudinal microtubules arrays. During IFM developing of D. melanogaster a microtubules array, with their associated proteins, as well as non-muscular myosin are also involved in nuclei arrangements forming rows in the syncytial myofibers, elongation and muscle shaping [20, 21, 39, 76,77,78,79,80,81]. At pupal stage, myosin and actin organize in premyofibrils with immature sarcomeres then, hypertrophic muscle growth happens, accompanied by mitochondria extensive growth and fusion [82, 83]. IFMs have a very high-energy requirements which are supplied by specialized mitochondria [50, 51, 82, 83]. Here we show that A. aegypti flight muscles undergo major changes in their structure in the pupa to teneral transition. Mitochondria proliferate in pupa and coalesce rendering giant mitochondria with tubular cristae during final steps of myogenesis up to the adult stage. In D. melanogaster IFM mitochondria has been described the genetic control of outer and inner membranes fusion [82, 83], and the identification of these genes is an interesting research perspective in A. aegypti. On the other hand, teneral mosquito’s sarcomere evolve to attain, in the adult, the definitive organized pseudocrystal myosin: actin structure, in a 1:6 relation, similar to those observed in the fly [