OA is a catecholamine that acts as a neuromodulator in invertebrates (David and Coulon 1985; Roeder 1999, 2002, 2005). The role of neuromodulators was explained by “orchestration hypothesis,” which assumed that neuromodulator release into specific neuropils configures neural commands to produce a coordinated network activity (Sombati and Hoyle 1984). OA mediates its function by binding to G protein-coupled receptors, which included cyclic AMP production or Ca2+ release. OA regulates many physiological processes including aggression, fight-flight response, circadian retime and locomotion (Riemensperger et al, 2005; Schwaerzel et al, 2003; Unoki et al, 2005; Zhou et al, 2008).
Each leg segment of the multi-jointed legs of adult insects contains a stereotyped arrangement of muscles. Contractions of these muscles through motor neurons control the coordination of locomotion. OA plays an important role in this process. In locusts, OA is delivered in time for enhancement of leg muscle contraction (Duch et al. 1999). In this study, we focused on the role played by octopaminergic receptors in balanced locomotion using RNAi of each receptor with three GAL4 drivers. Targeted RNAi-mediated knockdown using the tubulin-promoter GAL4 driver showed that oamb-deficient flies had the weakest performance in the negative geotaxis assay. The present study revealed significant impairment in negative geotaxis performance when oamb deficiency was directed to the muscular or nervous system. The walking speed of oamb-deficient flies was lower than that of control flies. Moreover, most of leg parts of oamb-deficient flies was smaller than those of control flies. These findings are interesting as they provide evidence that oamb acts in leg muscles as a neuromodulator/neurotransmitter that is important for normal leg muscle architecture and size and for the coordination of muscle contractions required for balanced locomotion. A possible explanation for these structural changes is based on the known physiological role of oamb mediated signalling that induces Ca2+ oscillations due to Ca2+ release from intracellular stores (Balfanz et al,2005). This reaction is controlled by kinase and phosphatase activities (Hoff et al, 2011). Furthermore, blocking phosphatase activity in oamb expressing cells completely abolished Ca2+ oscillations (LIT). In mammals, it has been shown that Ca2+-dependent pathways control muscle development (Damm and Egli, 2014). Lowering intracellular calcium levels inhibits the differentiation of skeletal myoblasts into mature myotubes (Porter et al, 2002). Moreover, it has been shown in mammals that adrenergic receptors signalling regulated myoblast differentiation (Saini et al, 2010; Church et al, 2014). This might explain, why the lack of oamb-mediated signalling events during development leads to the observed structural changes. The neuromodulatory role of OA in skeletal and visceral muscle contraction was reported before (Evans 1981). Oamb expression was reported in adult Drosophila leg muscles (El-Kholy et al. 2015). Oamb, the invertebrate counterpart of mammalian α-adrenergic receptor, is also expressed in the oviduct muscles of female insects and regulates their contraction by elevating the cytosolic Ca+2 level (Lee et al. 2003) and in the tracheal system (El-Kholy et al. 2015). OA through oamb regulates other physiological processes such the induction of insulin release from insulinproducing cells causing changes in the amount of daily sleep and changes in fat storage, leading to lean adult flies (Crocker and Sehgal 2008; Crocker et al. 2010; Erion et al. 2012; Luo et al. 2014; Li et al. 2016).
To verify the role of oamb in the formation of normal leg muscle architecture, leg muscles of male Drosophila were analysed using transmission electron microscopy. The results revealed that in oamb - deficient flies, the leg muscles displayed abnormal morphologies of sarcomeres, disorganised myofibrils and mitochondrial abnormalities. For these reasons, male Drosophila flies hypothesize to show severe defective locomotion behaviour. The sarcomeres and the entire myofibrils showed loss of striations and structure. The sarcoplasmic reticulum around each myofibril was noticeably disintegrated; consequently, division of the muscle fibres into myofibrils was indistinct. Moreover, the myofibrils broke down forming spaces in the muscle fibre. The thickness of myofibrils that could be observed in the leg muscles of oamb -deficient flies was significantly less than that the leg muscles of control flies.
Z-lines as anchoring structures for myofilaments dictate the final length of sarcomeres. Z-lines would be expected to participate in the organisation of myofilaments during the initial stages of myofibril assembly. The results of this study showed that Z-lines within the leg muscles of oamb -deficient flies were split into smaller fragments that were dispersed among the sarcomeres, causing loss of the normal muscle architecture and consequently impairment of the negative geotaxis performance of corresponding flies. Recently, Sujkowski et al. (2020) reported that oamb expression in Drosophila is required for adaptations in skeletal muscles in legs.
Mitochondria play an important role in the muscular system. In Drosophila, myofibers have a greater dependency on mitochondria and lipid oxidation and, thus, the numbers of healthy mitochondria can influence the capacity to maintain muscle mass and function. In skeletal muscles, mitochondria regulate energy haemostasis by producing ATPs required for muscle contraction through oxidative phosphorylation. In addition, mitochondria contribute to Ca+2 homeostasis (De Stefani et al. 2011; Eisner et al. 2014), redox signalling (Finkel 2011; Sena and Chandel 2012), release of pro-apoptotic factors (Frezza et al. 2006), synthesis of haeme molecule and regulation of nuclear gene expression (Picard et al. 2014; Chae et al. 2013). In this study, ultrastructural changes were detected in the shape of mitochondria within the leg muscles of oamb -deficient flies. The mitochondria were electron lucent, enlarged with an irregular contour or small with a rounded contour and others were absent from certain areas of the muscle fibre. These changes could be the reason behind the observed disorder of muscles.
Ultrastructure changes in mitochondria occur in muscle disorders (Kelley et al. 2002; Chen et al. 2010; Crane et al. 2010; Sao et al. 2010; Austin and St-Pierre 2012), particularly in muscular dystrophy, while in degenerating fibres, the number of mitochondria is reduced and they disappear from severely atrophied muscle fibres.
Taken together, the data presented in this study reveal that octopamine signalling via oamb plays an essential role in adult Drosophila movement ability as well as in normal leg muscle architecture formation and interorgan communication between the nervous system commands to motor neurons and muscular tissue responses. It became apparent that information regarding biogenic amine receptors is very important form many points of view including comparative, evolutionary physiology and biochemistry as well as serving as possible specific targets for insecticides.