Effect of Secondary Metabolite Crude of Metarhizum anisopliea Fungus on the Second Larval Stage of the Housefly Musca domestica

: The house flies Musca domestica )Diptera:musidae) are the primary carrier of many pathogens such as cholera, typhoid, anthrax


Introduction:
Housefly (Musca domestica) is the most common fly on the planet.More than 100 infectious diseases have been linked to houseflies in humans and animals 1 .Pathogens which are transferred by M. domestica may cause cholera, anthrax, food poisoning, typhoid, diarrhea and shigellosis 2 .House fly is considered as a wide separate and a dangerous insect due to its characteristics which are facultative diapauses characteristics such as broad distribution, and high reproductive capacity leading to the adaptation of various agroecological habitats and it has become a primary pest, through its vomits or excreta and legs, it acts as a mechanical vector for disease transmission through contaminated water and food 3 The common insecticides pyrethroids, spinosad, pyrethrins, dichlorvos, imidacloprid and cyantraniliprole, are used in house fly control 4 .Due to the dangers and hazards of chemical pesticides accumulating in the environment and being transmit into food, which kill untargeted insects, the world has resorted to using alternative pesticides, such as physical and biological control.The biological repression of insect pests such as viruses, fungus, bacteria, protozoa, and nematodes is known as microbial control 5 .The aqueous and alcoholic extracts of plant were tested on the insect which achieve high mortality rates 6 .Even the use of alternative chemicals such as citric acid and bicarbonate have effects on mosquitoes larvae but their safety on the environment is uncertain 7 .Microbial biocontrol agents (MBCAs) have the ability to eliminate pest populations while show no risk to human health or the environment, as a substitute for chemical pesticides, this is a viable option 8 .This Entomopathogenic fungus (EPF) is thought to be eco friendly and capable of producing toxic compounds that can kill target insects like cyclopeptida destruxin A, B, C, D, and desmethyldestruxin B. The fungus Metarhizium anisopliae and Beauveria bassiana are two fungi that are known to be entomopathogenic 9 .The secondary metabolites of B. bassiana and Metarhizium anisopliae as (n-Hexadecanoic acid; 9, 12-Octadecadienoic acid, methyl ester, (E, E)-; Hexadecanoic acid, methyl ester; 7,10-Octadecadienoic acid, methyl ester and trans-13-Octadecenoic acid; Tetradecanoic acid, 12-methyl-, methyl ester),have an insecticidal activity and antifeeding properties against insect as well as they caused larval growth inhibition 10 .The development of biochemical weaponry like as enzymes, poisons, and other metabolites compounds aid in host infection and invasion.Chitinase, proteinase, and lipase are enzymes that are directly involved in the destruction of the host cuticle, which is the initial and most important barrier to EPF infection,destruxins of Metarhizium, beauvericins of Beauveria, hirsutellides of Hirsutella, isarolides of Isaria, cordyols of Cordyceps, vertihemipterins of Verticillium, and other secondary metabolites disable and accelerate the EPF infection process by directly and indirectly disabling the insect hosts' defense mechanisms, Secondary metabolites range in chemical complexity from simple non-peptide pigments like oosporine to very complex piperazine derivatives 6,11 .Topical application of crude extracts did not result in death, but combined treatments with fungal suspensions of M. anisopliae and their extracts resulted in higher mortality rates than solo M. anisopliae and extract treatments 12 .Some entomopathogens' secondary metabolites have been shown to be beneficial in IPM programs against a variety of insect pests 10 .The most common secondary metabolites are produced by M. anisopliae during fermentation and, by far, the most thoroughly studied entomopathogenic fungus poisons recognized as key virulence factors hastening the mortality of infected insects.There are 38 Dxs or Dx analogs, which are chemically grouped into five groups named A through E 13 .The insecticidal activities of destroxins against Diptera, leading to the suggestion that destruxins could be utilized as pesticides, these toxins weaken the host's immune system, harm the muscular system, and damage the malpighian tubules, altering excretion and causing eating and movement problems 14 .As a result, the destruxins' function of limiting host mobility would also weaken this behavioral defense mechanism.Indeed, isolates of Metarhizium that produce more destruxins are more virulent 15 .The purpose of this research is to identify specific toxin and secondary metabolites of the entomopathogenic fungi Metarhizium anisopliae in order to improve its insecticidal action, reduce housefly infestation in the environment, and reduce the disease caused by houseflies .

Materials and Methods: Chemicals
Materials used were methanol ,ethyl acetate (Thomas Baker, India) as a solvent and as cosurfactant, tween 80 (Thomas Baker, India) as surfactant, and deionized water.

Musca domestica collection and rearing
Musca domestica adults were collected from a farm in Baghdad in Iraq government by using arial net.The adults were placed in wooden cage with a dimension of 30×20×20 cm 3 covered with tulle and one lateral side modified the tulle to enter the hand for supplement food and clean the cage.The adults of housefly were 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/ 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 the nutrition of adults for obtaining eggs placed under the larval nutrition , while larval grown in plastic container 20 ml capacity containing (float fish diet) that consists 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 crushed and ,where 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 was used to feed larvae and adult taking care of change it all three days 16 .The cage was placed in the rearing wooden cages with a dimension of 30×20×20 cm 3 .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.

Mass culturing of Metarhizium.anisopliae :
The fungi Metarhizium anisopliae was taken from Agriculutral Research Dpartment\ Ministry of Agriculture.The fungus was grown on PDA plates at 25±5°C for 3-5 days, to optain pure culture, 5 g/ml of chloramphenicol was added to Potato Dextrose Agr (PDA) after sterilized as antibacterial agent to prevent growth bacteria 17 .

Production of fungal toxic proteins and secondary metabolits in Potato Dextrose Broth (PDB ) medium:
In order to produce the secondary metabolic compounds of fungus Metarhizum anisopliae , 1cm piece of fungus was taken from three-day old PDA grown cultures by borer and cultured in 500 mL of PDB medium which consist of 200 ml potato water and water 800 mL , 20 g sugar, and 500g amoxylin in conical flasks and were incubated at 25°C and humedity 80% in an incubatore with shaking in different time for a period of 10-12 days. 18,19,20 Csical solvent extraction method: The majority of isolation procedures still utilize simple extraction procedures with organic solvents of different polarity, 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.The extraction of crude secondary metabolites was cmpleted 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 methanol) 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.Deionized water and tween 80 were used to dissolve the crud extract 21 .

Determine toxines and secondary metabolits in crude extract of Metarhizum anisopliae :
To determine toxines and secondary metabolits in crude extract of Metarhizum anisopliae used technique ( Gas Chromtography -Mass Spectrophotometery) ( GC-MS) .A Clarus 680 30 m × 0.25 mm ID × 250 μm silica column was used for GC analysis of the chemical constituents.This column was packed with elite-5MS 5% biphenyl 95% dimethylpolysiloxane.The chemical constituents were separated by using He at a constant flow of 1 mL/min as a carrier gas.The crude extracts (1 μL) were injected to the GC-MS instrument at 260 °C during the column running time.The temperature ramp was as follows: 60 °C (2 min); followed by 300 °C at the rate of 10 °C min−1; and finally 300 °C, where it was held for 6 min.The mass detector conditions were 240 °C; ion source temperature at 240 °C; and ionization mode electron impact at 70 eV, a scan time 0.2 s and scan interval of 0.1 s.The fragments from 40 to 600 Da were collected) 22 .

Bioassay of crud extract of M. anisopliae against the 2 nd larval stage of Musca domestica:
Experiment was conducted to bioassay of crud extract of M. anisopliae against 2 nd larval stage of Musca domestica ( diagnosis by head capsule ) by dissolving the methanolic crude extract in de ionized water and tween 80 to separate and dissolving the compound .The bioassay has three testing concentrations 1, 3, and 5% and each concentration had three replicates of 30 larvae in each concentration (10 larvae to each replicate).

Direct spray technique:
90 of 2 nd larval stage from culture divided to 3 replicates to each concentration( 10 larvae to each replicate ) ,spray it with the concentration 1 , 3,and 5% attended previously each separately ,then put the treated larvae on non treated medium cages that put in an incubator at 25±5 ⁰ C , 70% RH .Larval mortality rate was recorded after 24, 48, and 72h.

Indirect technique (treated rearing medium):
5 g of medium was used to rear Musca domestica put in container 50 ml , sprayed medium and container put un treated 2 nd larval stage .As 3 replicates of each concentration 1, 3, and 5%, 30 larvae in each concentration (10 larvae to each replicate), the experiment was put in an incubator at 25±5⁰C, humidity 70%.The mortality larvae was isolated and reported daily for 27 h.

Dipping technique:
In dipping method , 90 of 2 nd larval stage was divided into 3 replicates of each concentration 1, 3,and 5%) dipping 30 larvae with 1ml of each concentration for 1 minute each separately ,then transfer to non treated medium cage with smooth brush ( 10 larvae to each replicate ).Then, the cages were put in an incubator at 25±5⁰C humidity 70%.The isolated and reported larval mortality rate was recorded after 72 hours.

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.The lethal and sub-lethal concentrations were determined by a probit analysis software program

Determining toxines and secondary metabolits in crude extract of Metarhizum anisopliae : GCmass technique ( Gas Chromtography -Mass Spectrophotometer) Analysis:
GasChromatography -Mass Spectrometry results obtained from M.anisopliae indicated the presence of several major compounds (secondary metabolism) , as shown in Table 1 and Figure

Bioassay of M. anisopliae extract in the 2 end larval stage of housefly:
The results in Table 2 show the effect of concentration 1,3,and 5% with 3 techniques as, in direct spray technique in the concentration 1% were 0.0 , 0.0 , and 6.67 % mortality respectively after 24, 48 , and 72 hours of treatment .On the other hand, in the concentration 3% the mortality rate was 23.3 , 40.0 , 60.0 % respectively after 24, 48 ,72 hours .In the concentration 5% mortality rate was 63.3 ,73.33 , 73.33 % respectively after 24, 48 , and 72 hours of treatment .Statistical analysis reported a significant difference in mortality percentage with increasing time in rate 2.22 , 41.11 , 70 respectively , the concentration 3 % was the best to obtain higher rate mortality at LSD 0.05 .As the results in Table 2 record, the lowest mortality rates were 0.0 , 23.3 , 63.3 in the concentration 1, 3, and 5% after 24 hour of treatment , and the highest mortality rates were 6.67 ,60, 73.33 % in concentration 5% after 72 h , at LSD 0.05 showed a significant difference between concentration and the important interaction between treatment factors by increasing larval mortality with increasing time of exposure to extract and concentration .
Theirwas a significant difference at LSD 0.05.The results showed the interaction effect between treatment factors had significant effect on the increasing mortality rate with significant difference, as well.
In dipping technique , the results showed the mortality percentage in concentration 1% was 10, 10, 10 % respectively after 72 hours, while in concentration 3 , and 5% the rate mortality was 0.0, 0.0.100 % after 72 hours of treatment.Statistical analysis showed the significant effect of the treatment factors interaction.The result in rearing medium only technique showed the difference between application methods as high effect was with sprayed both rearing cage and larvae with nutrient medium in statistical analysis at LSD 0.05.
As statistical analysis of factors experiment show that the concentration 3% was the best.
Results in Table 3 showed sub lethal and median lethal concentration under different approach to control population of housefly, treated rearing cage more effect with LC50 was 0.26 , which maybe returned to increase the exposure to the toxic by treating rearing cage with nutrient media contain larvae , while lower LC50 was in dipping technique as 0.04.This agrees with the results in Table 2.
Treated larvae with 1, 3, and 5 % concentration of M. anisopliae required 72 hours to reach the mortality rate of 100% of methanolic extract crud ( secondary metabolites ) of fungus M. anisopliae, while it reported their significant difference between concentrations ,and the concentration 5% gave the highest mortality after 72 hours.Infected larva manifested numerous symptoms of unregulated movement, anti-feeding, change color from wax to brown then black and no development in growth larvae and not reaching the pupa stage.1%, 3% low concentration have fatal attracting effect while high concentration 5% has a repellent and anti-nutritive effect.The differences between application methods have high effect, sprayed both rearing cage and larvae with treated medium, which gave more chance to exposure the larvae to the second compound.The result agrees with (Ortiz , 2010).Testing the crude extract offers an alternative approach and is recommended when assessing the risks of metabolites for registration purposes.
The toxicological risk evaluation of metabolites produced by the insect pathogenic fungus Metarhizium anisopliae was spurred by growing sensitivity to secondary metabolites from fungal biological control agents (BCAs), Individual metabolites (destruxins A, B, and E) as well as the whole crude extract from liquid cultures were used to compare the two ways of assessing individual metabolites (destruxins A, B, and E) or the complete crude extract from liquid cultures.The crude extract contains synergistic and antagonistic actions.The findings show that identifying and assessing each unique metabolite produced by a BCA is not only time and money consuming, but also does not provide a complete picture.When analyzing the risks of metabolites for registration reasons, testing the crude extract is an alternate approach that is advised 28 .Several entomopathogen-secreted secondary metabolites have been reported to exhibit anti feeding and insecticidal effects 29,30 .
Also, it might be due to the presence of an endogenous lipid layer in M. anisopliae.Above all, ecdysis is a physiological mechanism that eliminates increasing infections as well as the old cuticle, boosting the chances of the host surviving 31 .Immune modulator secretion and protease repressor secretion are two primary methods by which an invading pathogen overcomes host immune defenses, M. anisopliae produced a collagen-like immune evasion protein that served as an antiadhesive protective covering, masking antigenic cell wall-glucans and prevented haemocytes from recognizing the hyphal bodies 32 .They're cyclopeptides and cyclodepsipeptides that are physiologically active and cause direct cytotoxicity 33 .
Increasing sensitivity towards secondary metabolites from fungal biological control agents (BCAs) has prompted the toxicological risk assessment of metabolites produced by the insect pathogenic fungus Metarhizium anisopliae.Main destruxins A, B and E. Evaluation of the cytotoxic activity of these different compounds suggested that a wide range of metabolites with synergistic effects are present in the crude extract , These metabolites also play a role in antimicrobial peptide synthesis and phagocytosis resistance.Destruxins from Metarhizium, on the other hand, are implicated in the creation of oxidative stress, which causes many of the host antioxidant enzymes to be destroyed 34 .However, one drawback to using fungi to manage arthropods is that they may kill their hosts too slowly.These toxins weaken the host's immune system, harm the muscular system, and damage the Malpighian tubules, altering excretion and causing eating and movement problems 14 ..As a result, the destruxins' function of limiting host mobility would also weaken this behavioral defense mechanism.Indeed, isolates of Metarhizium that produce more destruxins are more virulent 15 .

Conclusion:
It is believed that methanolic crude extract (secondary metabolites and toxin) of fungus Metarhizium anisopliae has a natural pesticide activity.It can be concluded that methanolic 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.

Authors' declaration:
- Fungi M. anisopliae characterized with radial growth and green cray color and seriales of elongated sporeses, Fig 1 (a,b,c).a ) serial boards spores 40X .( b ) radical growth of Metarhizium anisopliae in PDA ( c ) growth fungus in PDB .crude extract of Metarhizum anisopliae : Crude extract of Metarhizum anisopliae obained was yallow -riddish and gellatenouse in nature texture with bad smell , figure 2 .

Figure 2 .
Figure 2. Gelatinouse nature of crud extract of M. anisopliae after drying.

Table 3 . Lethal and Sub lethal dose of toxicity of M. anisopliae derived secondary metabolites on 2 nd larvae of Musca domestica under different application methods.
Conflicts of Interest: None.-We hereby confirm that all the Figures and Tables in the manuscript are mine ours.Besides, the Figures and images, which are not mine ours, have been given the permission for re-publication attached with the manuscript.-The author has signed an animal welfare statement.-Ethical Clearance: The project was approved by the local ethical committee in University of Baghdad.