Introduction

Locomotor activity is an integrative characteristic of the functional state of the nervous system as it is implicated directly or indirectly in most types of insect behaviours such as foraging and mating. Distinct brain structures like the mushroom bodies or the central complex have been shown to be required for the control of locomotor activity1,2. In adult Drosophila, locomotor activity can be measured by different tools. Negative geotaxis performance is an innate escape behaviour in which adult flies ascend food vials as a response to tapping the vials. It could be used as a tool to evaluate the locomotor activity of Drosophila. This behaviour requires nervous system signaling commands via different biogenic amines.

Octopamine (OA) is one of these biogenic amines that acts as a neurohormone, neurotransmitter and neuromodulator in invertebrates. This catecholamine was first discovered in the salivary glands of Octopus vulgaris3. OA is synthesised from the amino acid tyrosine. Tyrosine is converted by decarboxylation to tyramine by the enzyme tyrosine decarboxylase, and then tyramine is converted by hydroxylation to OA with the help of tyramine beta-hydroxylase. OA orchestrates many diverse physiological and behavioural processes including aggression, fight-flight response, circadian rhythm and locomotion4. OA is essential for modulating the function of skeletal and visceral muscles5. OA in invertebrates has the same function of noradrenaline in vertebrates. OA exerts these functions by binding to and activating OA receptors belonging to the G protein-coupled receptor superfamily. Drosophila possesses four OA receptors: Octß1R (also referred as oa2: CG6919 ), Octß2R (CG33976, CG6989) and Octß3R (CG42244, CG7078) and OctαR (also referred as oamb, CG3856)6. OctαR has two isoforms differ in the 3rd cytoplasmic loop and downstream sequence due to splicing of the last exon, both isoforms are highly enriched in mushroom bodies7. OA stimulates glycogenolysis, modifies muscle contraction, supports long-term flight and regulates “arousal” in the central nervous system8. Despite the potential relevance of OA for controlling locomotion, our knowledge about the role of each octopaminergic receptor still limited.

The GAL4/UAS system enables scientists to study either gain-of-function or loss-of-function of the gene (s) of interest9, facilitating studying gene expression in Drosophila10. The GAL4/UAS system has been used in RNA interference (RNAi) technology. This technology enables the knocking down a specific gene of interest.

The main goal of this work was to reveal which one of the Drosophila OA receptors is important for adult locomotion. We achieved this by knocking down each OA receptor everywhere within the insect body using a universal driver and performing the negative geotaxis assay with the corresponding adults. In order to explore the underlying mechanisms by which OA signalling controls locomotion, we asked whether the role of OA originates from its effect on the nervous system, more specifically from motor neurons or mechanosensory neurons sensing the gravity or muscular system. To address this point, specific GAL4 drivers were crossed to OA receptor (s) followed by evaluation of the climbing ability performance of F1 male adults. Finally, we examined the morphometric and skeletal muscle ultrastructure changes in the legs of adult flies that displayed the knocking down of OA receptors. The findings of the current study provide evidence that OA via OctαR orchestrates nervous system commands with muscular system response, consequently controlling balanced locomotion and that the underlying mechanism by which OA controlling locomotion is a multifactorial complex process more than anticipated. Moreover, the current study is the first that link the leg muscles development with neurohormonal control. Information regarding biogenic amine receptors is very important for comparative, evolutionary physiology and biochemistry as well as serving as possible specific targets for insecticides.

Results

In order to establish an overview of negative geotaxis performance relevant to OA receptors deficiency, RNAi lines targeted against the OA receptors were crossed with strong ubiquitous driver line tubP-GAL4 which was reported before to cause high percentage depletion in the expression level of target gene11 in most organs of the fly. In this study, we used RNAi construct (#31171) targeting both octαR isoforms. The level of RNAi-based knock down of OA receptors in F1 adults was validated using qRT PCR (Fig. 1). Results showed significantly strong KD with low leaky expression (Student t-test: t-value was 8.82, 9.03, 11.28 and 10.74 for octβ1R, octβ2R, octβ3R and octαR, respectively and P value was 0.001 for octβ1R, octβ2R and < 0.001 for octβ3R and octαR). Negative geotaxis assay results obtained revealed that despite all receptors receptive for OA have significant adverse effect on climbing performance, OctαR-deficient flies showed the most severe impairment, which was highly significantly different (P = 0.005) from that of control flies (Fig. 2). The control male flies showed the native characteristic strait-line walking behaviour to climb the cylinder. By contrast, OctαR-deficient male flies showed impairment in moving up against gravity. Some flies attempted several times to climb up the cylinder for a short distance, but they failed to maintain their equilibrium and fell into the bottom of the cylinder. Furthermore, few flies showed complete defective locomotion behaviour. They remained on the cylinder bottom walking very slow throughout the experiment. The same climbing impairment was observed using KO lines (Fig. 3). Therefore, we focused on whether the underlying mechanism of octopamine signalling via OctαR in climbing performance originated from the nervous system or muscular system. To achieve this, the OctαR RNAi line was crossed to two driver lines elav-GAL4 and mhc-GAL4 lines, respectively. The results showed that targeting the RNAi effect of OctαR either to the nervous system (df = 28, t = 10.396, P ˂ 0.001) or muscular system significantly (df = 28, t = 6.401, P ˂ 0.001) decreased the climbing ability of adult males compared with control flies (Fig. 4A,B). More specifically, when OctαR knocked down exclusively in motor neurons-specific manner using GAL4 driver, D42-GAL4, the same impaired climbing behavior was observed (Fig. 4C).

Figure 1
figure 1

Use of qPCR to validate knock-down of octopaminergic receptors genes. Relative level of expression of octβ1R, octβ2R, octβ3R and octαR in knocked down vs control group. Mean ± SD; N = 3; **P ˂0.01, ***P ˂0.001.

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Figure 2
figure 2

Climbing speed of adult Drosophila males that lost the expression of four different receptors receptive for octopamine using ubiquitous driver line tubP-GAL4 as compared to controls. The experiment was performed in triplicate. Mean ± SD; N = 10; *P ˂0.05, **P ˂0.01, ***P ˂0.001.

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Figure 3
figure 3

Climbing speed of male flies depleted of OA receptors. Three asterisks indicate high significance (Student t-test: P ˂ 0.001).

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Figure 4
figure 4

The climbing speed of OctαR-deficient adult Drosophila males (N = 10) as compared to controls. The experiment was performed in triplicate. RNAi-mediated gene kockdown was directed to the nervous system using elav-GAL4 driver (A), muscular system using mhc-GAL4 driver line (B) and to motor neurons using D42-GAL4 driver (C). Three asterisks indicate high significance (Student t-test: P ˂ 0.001).

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As sensing the gravity through feedback of mechanoreceptors is also essential for balanced climbing behavior, we asked whether OctαR involved in this signal. To address this question, we knocked down octαR using two mechanoreceptor GAL4 drivers, pain-GAL4 and iav-GAL4. In addition, we used another driver line R74C07-GAL4 labelling mechanosensory neurons in parts of the body (eyes and posterior abdomen) other than antennae12. Results showed significant reduction in climbing performance in response to manipulation of OctαR in mechano-sensing manner (Fig. 5).

Figure 5
figure 5

Negative geotaxis assay of adult male flies (N = 10) that lost the expression of octαR using specific mechanoreception GAL4 driver lines. Asterisks indicate significant differences among treatments (Student t-test).

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To investigate whether knocking down OctαR specifically in the muscular system cause morphometric changes in adult leg parts, the length and width of fore and hind leg segments were measured (Fig. 6). The results indicated that the lengths of all parts of forelegs (t = 8.013, P = 0.001 and t = 5.433, P = 0.006 for coxa and trochanter, respectively; t = 15.427, P = 0.000 for femur; t = 18.933, P = 0.000 for tibia and t = 22.232, P = 0.000 for tarsi) and hind legs (t = 16.324, P = 0.000 for coxa, t = 22.167, P = 0.000, for trochanter; t = 13.050, P = 0.000 for femur; t = 2.848, P = 0.046 for tibia and t = 2.842, P = 0.047 for tarsi) of OctαR-deficient flies were significantly different from those of control flies. In addition, the width of the trochanter and femur of the forelegs (t = 11.954, P = 0.000 for trochanter and t = 7.139, P = 0.002 for femur). In addition, the width of coxa (t = 14,613, P = 0.000), trochanter (t = 7.669, P = 0.002) and femur (t = 3.238, P = 0.032) of the hind legs were significantly different from those of control flies. In addition, OctαR knock out mutant flies which showed significant reduction in fore and hind leg segments as compared to those of w1118 flies (Fig. 7).

Figure 6
figure 6

Length and Width of fore and hind legs' parts of adult flies that lost the expression of octαR in muscular system as well as matching control adult Drosophila males. Mean ± SD; N = 10; *P ˂0.05, **P ˂0.01, ***P ˂0.001.

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Figure 7
figure 7

The effect of OctαR knock out on the length and width of fore and hind legs' parts as compared to those of w1118. Mean ± SD; N = 10; *P ˂0.05, **P ˂0.01, ***P ˂0.001.

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In this study, ultrastructure examination of femur region of the metathoracic leg of control flies revealed that the tubular oblique muscles consist of spindle-shaped myofibrils, arranged in parallel and had the known striated pattern of successive sarcomeres (Fig. 8A,B). All muscle fibres were ensheathed in a sarcolemma. The sarcolemma invaginates into fibres at regular distances. Each sarcomere extends between adjacent Z-discs of the myofibril. The Z line generally looks like a series of dense bodies and disconnected through the sarcomere. Their roundish mitochondria are present between the myofilaments and contain an electron-dense matrix. When OctαR knocked down in the muscular system using mhc-GAL4, the muscles displayed disorganisation of all components. The sarcomeres and the entire myofibrils showed loss of striations and structure (Fig. 8C,D). The sarcoplasmic reticulum around each myofibril was noticeably disintegrated such that division of the muscle fibres into myofibrils was indistinct. The myofibrils broke down such that there were spaces in the muscle fibre. The Z line split into smaller fragments that were dispersed among the sarcomeres. Although mitochondria were distributed along damaged myofibrils, some were enlarged with an irregular contour, some were small with a rounded contour and others were absent from certain areas of the muscle fibre. Their cristae were electron lucent.

Figure 8
figure 8

The effect of OctαR knocking down in the muscular system on the ultrastructure of the muscular tissue of the femur region of the hind legs as well as matching controls using mascular system driver, mhc-GAL4. (A,B) TEM micrograph of male flies of the controls of RNAi experiment. The muscles display features typical of insect skeletal muscle. (C,D) TEM micrograph of OctαR deficient flies leg muscles. M mitochondria, Myo myofibrils, Z z-line.

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Discussion

OA is a catecholamine that acts as a neuromodulator in invertebrates4,13,14,15. 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 activity16. 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 locomotion17,18,19,20.

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 contraction21. In this study, we focused on the role played by octopaminergic receptors in balanced locomotion using RNAi of each receptor with different GAL4 drivers. Targeted RNAi-mediated knockdown using the tubulin-promoter GAL4 driver showed that OctαR-deficient flies had the weakest performance in the negative geotaxis assay. This result was confirmed by evaluating climbing performance in OA receptors knocked out (KO) mutant lines which was lower than wild type w1118. When OctαR deficiency was directed to the muscular, nervous system and more specifically motor neurons, we observed significant impairment in negative geotaxis performance. The walking speed of OctαR-deficient flies was lower than that of control flies. Moreover, most of leg parts of OctαR-deficient flies was smaller than those of control flies. Smaller leg parts was also observed in OctαR-kocked out adults as compared to w1118.

Sense gravity via mechanosensory structure located in the antenna is important for spatial orientation for gravity negative geotaxis22. Many genes are involved in this process such as painless (pain)23 and inactive (iav)24,25 which are expressed in Johnston’s organ chordotonal neurons and femoral chordotonal organs and is required for normal hearing as well as gravity sensing22. We asked whether octαR play role in orchestration of feedback signals received via mechanosensory receptors required for normal climbing behavior response. To address this point, we knocked down octαR in two gravity-sensing manner using mechanoreceptor GAL4 driver line namely pain-GAL4 and iav-GAL4. In addition, we used another driver line R74C07-GAL4 labelling mechanosensory neurons in parts of the body (eyes and posterior abdomen) other than antennae12. Results showed reduction in climbing performance in response to manipulation of OctαR in mechano-sensing manner which may provide more additional underlying mechanism of OA signalling control of climbing behaviour via OctαR. It was reported before that OA increased responses of mechanically stimuli26,27,28. Torkkeli et al.29 stated that the OA receptor expressed in mushroom bodies increases the sensitivity in the spider Cupiennius salei mechanosensory neurons. These findings are interesting as they provide evidence that OctαR acts in leg muscles as a neuromodulator/neurotransmitter that is important for the development of normal leg muscle architecture and size and for the coordination of mechanical stimuli sensation with muscle response which is required for balanced locomotion. A possible explanation for these changes is based on the known physiological role of OctαR mediated signalling that induces Ca2+ oscillations due to Ca2+ release from intracellular stores30. This reaction is controlled by kinase and phosphatase activities31. Furthermore, blocking phosphatase activity in OctαR expressing cells completely abolished Ca2+ oscillations (LIT). In mammals, it has been shown that Ca2+-dependent pathways control muscle development32. Lowering intracellular calcium levels inhibits the differentiation of skeletal myoblasts into mature myotubes33. Moreover, it has been shown in mammals that adrenergic receptors signalling regulated myoblast differentiation34,35. This might explain, why the lack of OctαR-mediated signalling events during development leads to the observed structural changes. The neuromodulatory role of OA in skeletal and visceral muscle contraction was reported before36. OctαR expression was reported in adult Drosophila leg muscles37. OctαR, 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 level38 and in the tracheal system37. OA through OctαR regulates other physiological processes such the induction of insulin release from insulin-producing cells causing changes in the amount of daily sleep and changes in fat storage, leading to lean adult flies39,40.

To verify the role of OctαR in the formation of normal leg muscle architecture, leg muscles of male Drosophila were analysed using transmission electron microscopy. The results revealed that in OctαR-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 OctαR-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 OctαR-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.44 reported that OctαR 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 homeostasis45,46 redox signalling47,48, release of pro-apoptotic factors49, synthesis of haeme molecule and regulation of nuclear gene expression50,51. In this study, ultrastructural changes were detected in the shape of mitochondria within the leg muscles of OctαR-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 disorders52,53,54,55, 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 OctαR 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.

Contribution to the field

Biogenic amines act as neurohormones, neuromodulators and neurotransmitters controlling many physiological processes in both vertebrates and invertebrates. Some commercially available pesticides act on biogenic amines such as Rotenone, neonicotinoids and formamidine. Biogenic amines-based pesticides have adverse effects on human, especially nervous system causing neurological diseases. For this reason, the biogenic amine octopamine and its related receptors represent good insecticide targets as being exclusive to invertebrates. Consequently, detailed information about the contribution of single receptor in vital physiological and behavioral processes may help to choose the most effective target for specific insecticides design tools. In this study, we focus on the contribution of octopaminergic receptors in locomotion, as a crucial behavior for survival. Results of tissue specific RNAi-based experiments revealed that OctαR-deficient flies showed impaired negative geotaxis and leg muscles atrophy. In addition, this study revealed for the first time, the role of OctαR in the development of normal leg muscles architecture. Previous studies stated that agonists and antagonists for octopamine receptors act as potential targets for insecticides56, here our results specify OA receptor OctαR as a possible target for molecular docking for synthesis of specific insecticides due to the essential role of this receptor in locomotion and skeletal muscle development. In addition to this, the data provide new insights into what may be a potential future prospect for human muscle atrophy therapeutics by targeting the α-adrenergic receptors, the counterparts of invertebrate OctαR.

Materials and methods

Drosophila strains and rearing

All Drosophila lines (Table 1) were obtained from Bloomington Drosophila Stock Center (Bloomington Stock Centre, Indiana University, Bloomington, USA). These lines included RNAi effector lines generated by the Transgenic RNAi Project for the knockdown of Drosophila OA receptors (RNAi for DmOctαR#31171, DmOctβ1R# 31106, DmOctβ2R# 34673 and DmOctβ3R# 31108) and GAL4 driver lines (tubP-GAL4 #5138, elav-GAL4 # 8765, Mhc-GAL4#55133, pain GAL4 # 27894, D42-GAL4 #8816, iav-GAL4 #52273 and R74C07-GAL4 (meru-GAL4) # 39847). First, the efficiency of knocking down of RNAi transgenes was validated using qRT PCR as described before57. Primer sequences are as follow: DmOctαR forward 5ʹ-CGG TTA ACG CCA GCA AGT G-3 ʹ; DmOctαR reverse 5ʹ-AAG CTG CAC GAA ATA GCT GC-3ʹ, DmOctβ1R forward 5ʹ-GGC AAC GAG TAA CGG TTT GG-3ʹ; DmOctβ1R reverse 5ʹ-TCA TGG TAA TGG TCA CGG GC-3ʹ, DmOctβ2R forward 5ʹ-TCC TGT GGT ACA CAC TCT CCA-3 ʹ; DmOctβ2R reverse 5ʹ-CCA CCA ATT GCA GAA CAG GC-3ʹ, DmOctβ3R forward 5ʹ-TGT GGT CAA CAA GGC CTA CG-3ʹ; DmOctβ3R reverse 5ʹ-GTG TTC GGC GCT GTT AAG GA-3ʹ, and a house keeping gene rpl32 forward 5ʹ-CCG CTT CAA GGG ACA GTA TC-3ʹ; rpl32 reverse 5ʹ-GAC AAT CTC CTT GCG CTT CT-3ʹ. Then, for knocking down octopamine receptors, virgin females carrying GAL4 driver was crossed with: (i) UAS-RNAi effector lines males (ii) W1118 (#3605) males to produce offspring as the first control for RNAi experiment. Additionally, W1118 virgin females were crossed with males carrying RNAi transgenes to serve as the second control for RNAi experiment. At least two available constructs for the same receptor-coding gene have been tested to be sure the observed phenotype is not due to off-target effects and the results presented in the manuscript were to the strongest construct which show high significant difference. Drosophila mutants for octopamine receptors (DmOctαR KO#83030, DmOctβ1R KO# 83035, DmOctβ2R KO# 82785 and DmOctβ3R KO# 83046) were obtained from Bloomington Drosophila Stock Center. All lines were reared on standard Drosophila medium (14–15 g agar, 18.5 g yeast, 61 g glucose, 30.5 g sucrose, 101 g corn meal/L) at 25 °C except for F1 of the crossings was kept at 27–29 °C to enhance the RNA interference effect. All lines were kept at 50%-60% relative humidity with an 18/6-h light/dark cycle.

Table 1 Stock number, name, genotype and usage of Drosophila lines used in this study.
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Climbing assay (negative geotaxis)

A negative geotaxis assay was performed using only male adult flies to avoid the effect of weight changes in adult female flies due to the physiological process including ovulation and oogenesis. Briefly, 10 males were placed into a 100 mL empty glass cylinder. The flies were tapped to the bottom of the cylinder and left to climb the cylinder. This procedure was repeated five times. The upward movement of the flies was recorded until the last climbing fly reached the top of the vial. The upward movement of flies was recorded with a digital video camera. The videos were cut into snapshots at the rate of 5 frames/second. These frames were analysed using ImageJ 1.46r software (National Institutes of Health, Bethesda, MD, USA) to trace the upward paths of the flies. The walking speed was calculated by dividing the length of the path (cm) by the time required in each path (s)58,59.

Effect of OA receptor knockdown on climbing ability

OA receptors were silenced using the GAL4/UAS system. First, the universal driver tubulin promoter GAL4 (tubP-GAL4), which induces gene repression in almost all insect’s body cells, was independently crossed to the UAS-OctaR, Octß1R, Octß2R or Octß3R RNAi effector lines to detect the OA receptor which is strongly involved in climbing performance. The climbing ability of ten F1 adult males was studied as described above. Adult F1 males of crossing between w1118 and : (i) tubP-GAL4 and (ii) RNAi lines were used as controls. Then to explore the root cause of the behaviour performance impairment, we down regulate OctaR using specific tissue driver lines: nervous system-specific driver elav-GAL4, motorneurons driver D42-GAL4, muscular system-specific driver myosin heavy chain (Mhc-GAL4), gravity-sensing drivers Pain-GAL4, iav-GAL422 and meru-GAL4. GAL4 driver line were crossed to UAS-OctaR RNAi effector line and the climbing performance of F1 males (N = 10) was evaluated as mentioned above. In addition, the effect of OA receptors ablation on climbing performance was investigated using Drosophila knocked out (KO) lines for OA receptors.

Morphometric analysis of adult legs

Fore and hind legs were removed by forceps from F1 of UAS-OctαR-dsRNAi × mhc-GAL4. Then transferred to glass slides using a fine brush for microscopic examination using an Olympus BX61 light microscope at magnification 2.5 × 10. Photographs were taken and the length and width of each leg segment (coxa, trochanter, femur, tibia and tarsi) were measured using ImageJ software. In parallel, F1 of w1118 crossed to: (i) OctαR-dsRNAi and (ii) GAL4-mhc was used as controls for RNAi experiment. Additionally, morphometric analysis of OctαR KO adult legs was done. W1118 was used as control for knock out (KO) experiment.

Transmission electron microscopy

Ten hind legs from UAS-OctαR-RNAi × mhc-GAL4 adult offspring were removed by forceps. Then, the legs were fixed in 5% glutaraldehyde in sodium cacodylate buffer (pH 7.4) for 12–24 h. The specimens were washed and fixed overnight in 15% buffered osmium tetroxide. Samples were incubated overnight in 0.5% aqueous uranyl acetate solution, then dehydrated, filtered and embedded in spur resin60. Semi-thin (1 μm) sections were stained with methylene blue Atur II and examined using an Olympus BX61 light microscope. Ultra-thin sections (60 nm) were cut using an ultramicrotome (Ultracut S, Leica), and stained with 2.5% uranyl acetate and lead citrate and were examined using JEOL, JEM 100-SX electron microscope.

Statistical analysis

Data represented as (Mean ± SD). Normality of the data was tested with the Kolmogorov-Smirnov test. Two sample t-test was used to compare between control and other groups. For all statistical tests p-value > 0.05 is considered not-significant. p-value < 0.05 is considered significant. p-value < 0.001 is considered highly significant. All data were analyzed using, Minitab software package version 19.0 and Microsoft Excel 365.