Effect of nanocapsules and extract of Metarhizium anisopliae in inhibiting acetylcholine esterase enzyme in Musca domestica larvae.

: M. d omestica is the most important insect that transmit pathogens for diseases in the world. The use of nanotechnology is eco-friendly method in control pests. The study aims to investigate the feasibility of bio-manufacturing nanocapsules of fungal secondary metabolites in order to improve the efficiency of metabolite and assess their inhibitory effect on the acetylcholine esterase enzyme in housefly larvae. An equal mixture of organic solvents, ethyl acetate and dichloromethane, was used to extract the metabolic products of the fungus M. anisopliae , (PEG4000) and chitosan was used in the preparation of nanocapsules. The results of the DLS granular size assay showed that the size of the extract particles and the size of the chitosan and (PEG 4000) nanocapsules were 610, 217 and 188 nm, respectively. The SEM images showed that the diameter of the extract and the nanocapsules chitosan and polyethylene glycol 4000 reached a rate 547.5, 17.8 and 26.2 nm, respectively. The FTIR showed that the extract of the second products of the fungus contains functional groups like: alkynes and alkenes, amines, carboxyl and aromatic groups, while the presence of groups of phenols, alcohol, amines, alkenes, and alkyl halides was recorded for nanocapsules of chitosan and PEG. The results showed that the extract of fungal metabolic and nanocapsules has an inhibitory effect on acetylcholinesterase enzyme and reached the highest inhibition rate 53.2 ,36.3,18.2% when treated with nanocapsules PEG at a concentration 500 ppm, extract of fungal metabolites at a concentration 50,000 ppm, chitosan nanocapsules at a concentration 500 ppm respectively. It is clear that acetylcholinesterase inhibition is one of the mechanisms of fungi metabolic action and the nanocapsules prepared from them .


Introduction:
The housefly, Musca domestica (Diptera: Muscidae), is the fly that transmits the most diseases worldwide. Houseflies have been linked to more than 100 infectious illnesses in both people and animals, the pathogens that M. domestica transmits have been linked to cholera, anthrax, food poisoning, typhoid, diarrhea, and shigellosis 1 . Due to their vast range, strong reproductive potential, and facultative diapause, houseflies are regarded as a distinct species and a hazard to humans. They have also become a major problem since they may spread illness through polluted water and food using their vomit or excrement and legs 2 . Chemical pesticides have been extraordinarily successful against agricultural pests and arthropods that are crucial to human health, Insecticides often used to control house flies include pyrethroids, spinosad, pyrethrins, dichlorvos, imidacloprid, and cyantraniliprole 3 . Alternative methods of chemical pesticides like aqueous and alcoholic extracts of Piper nigrum were tested on the third instar larvae of Culex pipiens which achieve high mortality rates 4 . The advantages of neurotoxin insecticides include their quick action, which stops crop damage right away, their numerous highly sensitive sites of action, which make even a small disruption potentially fatal, their lipophilic nature, which makes it easy to penetrate the lipoidal sheath, and their lack of a detoxification mechanism in nerves, which prolongs insecticidal activity. Because of these benefits, the majority of synthetic insecticides on the market today have neurotoxic properties, frequently via blocking acetylcholine esterases (carbamates) or impairing the operation of ion channels in nerve cell membranes (pyrethroids) 5 . Entomopathogenic fungi (EPF) create secondary metabolites that might be a source for the development of biopesticides and operate on other species, occasionally preventing growth, causing illness, and even death 6 . Pest management and the manufacture of pesticides both employ nanotechnology increasingly 7,8 . Crop productivity can be increased with the use of nanotechnology, which can also be used to create novel pest control methods 9,10 . Green synthesis techniques are one of the techniques used in nanotechnology to create nanocapsuls, nanoparticals, nanosuspensions and nanoemulsion 11 . These techniques have certain benefits over others since they are often easier and less expensive to carry out and don't involve harmful chemicals that might pollute the environment. Stable nanocapsules are created using green synthesis techniques using biomolecules from plants and microorganisms, such as proteins, enzymes, phenols, amines, and alkaloids 12 . Saied (Kouzegaran and Farhadi, 2017) that the toxicity of secondary metabolites against insects can be improved by adding synergist agents such as chitosan or Polyethylene glycol 4000 (PEG) 13 . Chitosan is a naturally occurring substance that is obtained from marine crustaceans and shrimp. It is a biopolymer with many uses due to its solubility, high stickiness in acetic solution, non-toxicity to mammals, reactive amino (-NH2) and hydroxyl (-OH) group composition, high biodegradability, and antimicrobial properties. Due to these qualities, it may be used in a variety of fields, including the biomedical sector, pest control, the food industry, and environmental pollution reduction 14 . Additionally, it is non-toxic to both humans and vertebrates, so it has a variety of uses 15 . Polyethylene glycol 4000 is a synthetic, hydrophilic, biocompatible polymer with widespread use in biomedical and other applications 16 . Nanoparticles (NP) prepared with chitosan and chitosan derivatives typically possess a positive surface charge and muco adhesive properties that can adhere to mucus membranes and release the drug payload in a sustained release manner 17 . This study aimed to prepare Polymeric nanocapsules of crude extract of entomopathogenic fungus Metarhizium anisopliae by chitosan and Polyethylene glycol 4000 and assay their inhibition effect on acetylcholine esterase enzyme in Musca domestica larvae.

Material and methods: Chemicals
Materials that were used, like, Di chloromethane (Thomas Baker, India) as a solvent and as co-surfactant, tween 80 (Thomas Baker, India) as surfactant, and deionized water, chitosan, sodium phosphate buffer (Na2HPO4, NaH2PO4), fast blue B salt, acetylcholine chloride, polyethylene glycol 4000, and chitosan

Musca domestica collection and rearing
Musca domestica adults were collected from a farm in Baghdad \ Ghzallia\ Iraq by using an arial net. The adults were placed in a wooden cage with a dimension of 30×20×20 cm 3 . that was covered with tulle and one lateral side modified the tulle to enter the hand for supplementing food and cleaning the cage. The adult of housefly was sent to the Natural History Museum of Iraq for identification, they were diagnosed as Musca domestica. The experiments were done in the laboratory of the Agricultural Research directorate ministry of science and technology in Al Twaitha ⁄ Baghdad ⁄ Iraq.
In the laboratory, plastic containers of 500 ml were filled with cotton webbed with 1:1 sugar and distilled water to nutrition adults to obtain egg , while larval grown in plastic containers of 20 ml capacity contained of (float fish diet) consisting of crude protein 28.0 % Min. , crude fat 4.0 % Min. and crude fiber 4.0 % Max in addition to amino acids, vitamins , minerals , after being crushed were taking 200 gm of (fish diet) mixed with 10 gm of dry yeast , 5gm of citric acid and dissolved in 100 ml of distilled water , the nutrient which is used to nutrition larvae and adults take care of changing it all three days 18 . The rearing cages were placed in the incubator under controlled conditions 27± 2 º C, 65± 5 RH and 10: 14 D/L photoperiod. This feeding is important for Musca domestica to get the energy needed for flight and other life activities.
-Crude extraction of toxin and secondary metabolism from fungi M. anisopliae : The fungus Metarhizium anisopliae was taken from the Agricultural Research Department\ Ministry of Agriculture. To obtain the secondary metabolic compounds of the fungus Metarhizum anisopliae, a 1 cm piece of the fungus was taken from 3 day old PDA grown cultures by borer and cultured in 500 mL of PDB medium, which consisted of (200 mL potato water and water, 800 mL, 20 g sugar, and 500 g amoxylin) in conical flasks and were incubated at 25°C and humidity 80% in an incubator in an incubate with shaking in different time for a period of 10-12 days 19 . Classical solvent extraction method: the majority of isolation procedures still utilize simple extraction procedures with organic solvents of different polarities, water and their mixtures; The method includes maceration. Following incubation, the fungus biomass was separated from the culturing medium with gauze (soft) cloth in order to use the broth portion, which contained toxin, secondary metabolism, and spores. completing the extraction of crude secondary metabolites with Whatman filter paper No.1 separated the light-yellow color component of the fungal mat, organic phase. , 500 ml broth, 750 ml solvent 1:1 ethyl acetate and dichloromethan (DCM) ) which were mixed with the fungal broth component for cold extraction with shaking for 2-3 days at 28 ⁰C. The solvent was separated from the water by separating funel, and the solvent was filtered using a micropore filter 0.2 mM to clear it. A vacuum evaporator at 80 °C was used to separate the combined solvent from concentrated extracts 20 . Deionized water and tween 80 were used to dissolve the crud extract .

Preperation of M. anisopliae extract -Chitosan Nanocapsules by Ion Gelation method:
In this technique the chitosan solution positively charged is dissolved in acetic acid 0.35% w/v prepared by dissolving chitosan powder in 1.5% acetic acid solution negatively charged with a stabilizing agent such as STPP (sodium tripolyphosphate) was added as a cross linker. then, a crud dichloromethane extract of M. anisopliae 2 g was added to 20 mL chitosan solution, the whole mixture was then stirred at room temperature on a magnetic stirrer (Rexim RP-1DN, Agile) for homogenous. The obtained homogenous slurry was added to a screw cap vial. The change in color of the solution from colorless to yellow gradually confirmed nanocapsule synthesis, keep it at 4 ⁰C until use. Chitosan nanoparticles are readily formed due to complexation between positive and negative charged species during mechanical stirring at room temperature, resulting in the separation of chitosan in spherical particles of different sizes and surface charges. Generally, 21,22,23 . Treated the crud extract of M. anisopliae, the solution M. anisopliae-chitosan, with an ultrasonic device (COMNI INTERNATIONAL, Homogenizer company) by inserting the device's beam into the solution and running it for 10 minutes, then repeating the process five times separately. Each treatment is stored in a dark screw container until used.

Preparations of crud extract of M. anisopliaepolyethylene glycol Nanocapsules
PEG 49.5g was dissolved at 65 ⁰C, added crud dichloromethane extract of M. anisopliae 0.5 g with continuously stirring by using a magnetic stirrer. The heating was continued throughout the addition process to obtain the nanocapsules put the solution in the freezer until stiffened, then grind it as powder of nanocapsules. The production of nanocapsuls for crud dichloromethane extract of M. anisopliaepolyethene glycol 4000 was powdered and reddish in color.

Scanning electron microscope (SEM) analysis:
To determine the structure shape and size of crude extract and nanocapsuls of M. anisopliae scanning electron microscopy, samples were processed and fixed by following the method of Wang et al. 24 . The images were captured under SU8010 (Hitachi, Japan) SEM operating at an accelerated voltage of 5.0 kV 25 . -Fourier transform infrared spectra (FTIR) measurements.
To determine the functional groups in the crud extract and nanocapsuls of M. anisopliae through FTIR analysis using MIR8035 FTIR spectrometer (Thermo Fisher, Bremen, Germany) 26 .
-Bioassay of chitosane, polyethylene glycol 400 nanocapsul of crud extract Metarhizium anisopliae on larval acetyl cholinesterase enzyme: Treated larvae 5 g of medium that was used to rear Musca domestica was put in container of 50 ml , sprayed from 30 cm distance with 50.000 ppm of crud extract Metarhizium anisopliae , 500 ppm crud extract M.anisopliaechitosane nanocapsule , and 500 ppm M. anisopliaepolyethylene glycol 4000 nanocapsule , then put the larval 2 nd , 3 rd ,4 th stages without treated as 3 rep / 30 larvae, put the experiment in an incubator at 25±5 ⁰ C / humidity 70% . for 24 -48 h. Enzyme assay Preparation of whole-body homogenate for enzymatic estimation, the larvae of M. domestica were washed thoroughly with distilled water and the extra water adhering to the larvae was cleared by using filter paper. Then, treated larvae were subjected to homogenization utilizing ice-cold sodium phosphate buffer pH 7.0, 20 mM with the help of Teflon hand homogenizer then centrifugation was done at 4500 run and 4 ͨ C for 30 min and resultant supernatant was used for the estimation of acetylcholine esterase. Solutions and glassware used for homogenization were kept at 4 ͨ C before use, and homogenates were also placed on ice until utilized for enzyme assays 27 .

Estimation of acetylcholine esterase activity:
In the 50 ml of enzyme solution, 50 ml of acetylcholine chloride 2.6 mM as a substrate and 1 ml of SBP (sodium phosphate buffer, pH 7.0, 20 mM) were added. It was incubator at 25 ͨ C for 5 mints. Then 400 ml of 0.3% Fast blue B salt was mixed to stop the reaction. Blank and sample were run through a spectrophotometer. Optical density OD was checked at 405 nm. The percentage inhibition of the enzyme activity by the test extracts was calculated as follows: % Enzyme inhibition = OD of Control larvae _ OD of treated larvae / OD of Control larvae x100 28 .

Statistical analysis
The experiments were carried out according to factorial experiments using a completely randomized design (CRD), and the differences between the means of the treatments were tested according to the value of the least significant difference at the probability level of 0.05. The results were analyzed by the statistical program Genstat 10 29 .  The results in Table 1 and Fig.6 showed the effectiveness of crude extract M. anisopliae , nanoparticals of crude extract M. anisopliaechitosane , and nanoparticals crude extract M. anisopliaepolyethylene glycol 4000 nanocapsuls on larval acetyl cholinesterase enzyme . enzyme inhibition was 53.2 % with nano capsuls of M. anisopliaepolyethylene glycol 4000 in concentration 500 ppm , 18.2 % with M. anisopliaechitosane nanocapsuls in a concentration 500 ppm , and 36.3 % in crude dichloromethane extract of M. anisopliae in a concentration 50.000 ppm With the significance different in % enzyme inhibition at the level of probability 0 .05 . as in shape 4 the treatment with crude extract of M. anisopliae -polyethylene glycol 4000 nanocapsuls was the higher inhibitor to enzyme acetylcholine esterase in larvae Musca domestica with significant effect. While the lowest inhibitor was with treatment by crude extract of M. anisopliae -chitosan nanocapsuls. Recently, Nanotechnology is increasingly being used in areas of pesticide production and pest management. fungi target their hosts by producing toxins and anti-insect secondary metabolites resulting in an array of effects ranging from paralysis to immune-suppression 30 . The fungus gradually weakened the enzyme activities as time progressed, indicating that the physiological attributes of the host were adversely affected 17 . FTIR results of synthesized crud extract of M. anisopliae -polyethelenglycol nanocapsules showed the presence of O-H , C≡C , N-H , and C-CL theses found in Alcohol / phenol , Alkyne, Amine complex, and Alkyl halides, which is similar to the FTIR profile of M.anisopliea-chitosan nanocapsules, explained by Namasivayam et al 31 . The changes in C≡C , C-F , and N-H stretching in both formats of nanocapsule green synthesis structural changes in response to the polyethylene glycol 4000, chitosan coating of secondary metabolites of fungal extraction 32 . FT-IR spectral investigations revealed the presence of carboxylic acid, amides, and phenol groups, Aromatics compound, Alkane , Alken , Alkyne , Ether , and Halid alkyl all of which could be involved in inhibition enzyme activity , this agrees with Vivekanandhan et al . 33 . which reported that The M. anisopliae derived chemical constituents are effective on targeted pests, pollution-free, targetspecific, and are an alternative chemical insecticide 34 . The average size of nanocapsules format plays a major role in effect speed effect to inhibit enzyme activity, which includes the average size of M. anisopliae -chitosan nanocapsules was 17. and SEM analysis explain the difference between crud extract and nano format of green synthesis. The smallest average size of M. anisopliaepolyethylene glycol 4000 nanocapsules and nanocapsules M.anisopliaechitosan gave it the ability to enter the body and rapidly reach the target sit. the enzyme Acetylcholinesterase (AChE) found in the synaptic cleft of insects, is responsible for the deactivation of the principal neurotransmitter acetylcholine (ACh) that transmits nerve impulses from one nerve cell to another or to involuntary muscles , it conceder as the target site for neurotoxic insecticides 35 . Similarly, previous studies showed that the metabolites of entomopathogenic fungi are founded in several chemical classes (phenols, alcohols, misc, carboxylic acids, aromatics, phosphor amide, and disulfides), which may be engaged in the mosquitocidal effects 33 . The decrease in AchE activities can be related to the mode of action of both agents, phenol, an alkaloid extracted from M. anisopliae which targets insect Ach receptors that turn off AchE production 36 . Due to the accumulation of ACh in the synaptic cleft because of inhibition AChE enzyme production leads to continuous nerve signaling, and finally to paralysis and death of the insect 37 .

Result and discussion
The neurotoxic activity of crude extract and Nano DEPA treated mosquito larvae was determined by AChE assay 38 . Also, secondary metabolites may cause membrane depolarization Yadav et al. 39 .
Opening calcium channel caused tetanic paralysis in the insects Samuels et al. 40 . Alkenes are excellent starting materials for organic synthesis as a result of the changeable reactivity of C꞊C bonds and the easy availability of many unfunctionalized alkenes. Direct regio-and/or enantio selective conversion of alkenes into functionalized compounds has immense potential for industrial applications 41 . Due to PEG characteristics often selected for adsorption or grafting applications to surfaces of nanocapsule, including electrical neutrality, significant spatial repulsion, and hydrophilicity. After PEGylation, a hydrophilic protective layer is formed around the nanocapsule, which increases several times for the blood circulation half-life of the particles through spatial repulsion rejection, Also, it is easy for nanocapsule to use PEG as a bridge to conjugate targeted ligands or peptides onto its hydroxy terminal, which can bind the corresponding overexpressed receptors on the surface of cells, to realize targeted drug delivery. PEG can improve the targeting delivery capability of nanocapsules, inhibit the removal of nanocapsule by the mononuclear phagocytic system MPS, and tune some physicochemical properties of nanocapsule , such as the mechanical properties of membranes, stability, and drug loading and release behavior , Molecular weight MW and PEG density are the key factors affecting the physicochemical and biological properties of nanocapsule 42 . Many natural products trigger very specific physiological responses in other organisms and in many cases bind to the receptors which have a remarkable complement, which means that the natural products may aid in an organism's survival in the absence of an immune system. AChE enzyme has a large but flexible structure consisting of 535 amino acids with a 20 Å deep and 5 Å wide narrow tunnel leading up to its active site , The tunnel, also known as the active-site gorge, consists of 14 flexible aromatic amino acid residues, These aromatic residues play essential roles in binding to the substrate. Cholinesterase inhibitors enhance cholinergic activity by inhibiting enzymes AChE and/or BuChE that hydrolyze ACh following synaptic release and, hence, prolong the activity of ACh molecules 43 .

Conclusion:
It is believed that M. anisopliaepolyethylene glycol4000 nanocapsules of DCM crude extract (secondary metabolites and toxin) of fungus Metarhizium anisopliae has a natural inhibition activity of acetylcholine esterase enzymatic activity in M. domestica (L.). It can be concluded that M. anisopliaepolyethylene glycol4000 nanocapsules of DCM crude extract (secondary metabolites and toxin) of fungus Metarhizium anisopliae can be used as a safe and effective alternative in the control of vector-borne diseases caused by Mucidae larvae. Toxicity