1. INTRODUCTION:

Musca domestica: belong to order Diptera and Muscidae Family, it look upon the important medical insects and most successful in spreading, especially Muscidae family, this insect consider the most numerous, prevalence in most areas to live close garbage of the human and animal, because their habits such as vomit, excrements, dissolute organic materials for nutrition ,then left their eggs, when it come down and visit several persons, flies transmits many of pathogens with its legs, hair of body and pillows of sticky legs area as well as caused by their habits in nutrition of the inconvenience and harassment of human beings and animals [1].

It is well known that the M. domestica from insects with complete metamorphosis, starting with egg stage and then larvae, pupae, and followed by the adult insect, which look for food attracting to richness decaying waste food, dead animals or their remnants and exudates even it feeding on infected wounds in human and animals and materials of food, especially the waste piles of manure stationed near rights whether in the countryside or the city so associated with the presence poor conditions of cleanness[2]. After that it feed on those articles and put their eggs in the same environment and prefer to place the container on the wet food decaying to put their eggs so as to hatch to creamy small larvae nourished on dissolved organic materials in the environment in which the eggs pass in three larval instars afterwards these instars, the larvae migrate to dry places for purpose entry in the pupal stage, and then the adults stage come out after rupture of the cast of the pupae to fly, feed, intercourse and then looking for a place to put eggs thus resume their life cycle [1],[3][4] were mentioned that The M. domestic larvae after the third larval instarmigrate the nourish environment can crawl up to 50 feet trying to find the driest and coldness places , because of that nourishment center is often high temperature result of generated heat resulting from movement, contention and friction of those larvae together and their vital effectiveness.

[5] Recorded that the third in star of larvae after the completion of their nourishment begins preparation to leave the nourishment destitute or place of wet food[5], often ,some studies indicate that leave be morning, There are many factors influencing the behavior of non-adult stages of M. domestica .[6]was indicated the impact of the different seasons on the color and place of the pupae insect may be different the color and place according to season, because of that the place of pupal stage is lag because of the different the type of predators, such as birds or small reptiles, mammals [7], also reported that the M. domestica larvae which were rearing in covered containers included manures of pork, that 70% of the larvae have migrated from the center these containers which included that material, most of the larvae came into pupae stage after 48 hours of migration, It also he showed that most larvae succeeded to get out of those closed containers depending on the oxygen and light source.

The scientific research, especially the genetic, get used to use fruit fly in most such genetic researches, but in the current study have Selected samples as M. domestica flies and its larvae in training on the possibility of extracting and isolating DNA because of this insect characterized with many characters, such as exist this insects in most environments, it is not devoid area of it over all and it exist in most of the times, also easy of rearing and nutrition it and feed on most types of food, therefore easy access to pure generations of it, as well as easy to distinguish the identification of M. domestica is regard the number one insect which known and separable to human since the past.[5].

What for holdings of these fly from mentioned characteristics to match the fruit fly as well as the short life cycle and abundance it for training of new researchers on how isolate DNA from M.domestica or it larvae, as well as to investigate the whereabouts of existing and generate that insect ,consequently know it life cycle in the nature and the nearby places of any person which increases the curiosity of the researcher to observe the behavior of its larvae in type and method of nutrition and the time of the end of the stage of nutrition by the medical importance of this insect and movement and migration of larvae and distances which it pass to find places to hide beneath or inside it and then stop the movement and entry in pupa stage, particularly researches that have dealt with the distances and places for larval stages and pupae of M. domestica These researches regard of appliances were few, if were compared with the many of researches which concern with it damages and methods of control it. This research came not for the purpose of control or elimination of M. domestica but the possibility of scientific advantage of access this insect to explain simplified method for extracting the DNA of larvae of M. domestica as well as report some observations on some habits and characters, such as the place of pupal stages which distinguish of those larvae that it belonging to that genus or species.

MATERIALS AND METHODS:

(1): Rearing the flies M. domestica:

The samples of M. domestica flies were obtained from one of the houses in the residential areas of Diwaniyah city and left those samples in wooden cages made especially for rearing flies, its dimensions 50 *50*50 cm. under the conditions of 36C,̊ a Rh of 27% covered one of its side with piece of fabric having aperture circular sleeve like allows to entrya hand while others insects did not allow exit or enter inside cages, and had nourished the flies inside the cage according to method of [8], petri's dishes had placed inside it which contained a quantity of dried milk with sugar and the river or rain water to feed the adults and put within the cage cardboard include manure or sterilized excrements of birds (600gm), with a quantity of powder yeast (20gm) and put other petri's dishes contained grafts fish (200gm) ,then had wetted with little of river water or rain to help feed the larvae of the flies[8] ,Large numbers of larvae were obtained using this method(Picture1) thus access to sustainable household and generations of M.domestica flies

Picture (1): shows a large numbers of larvae of M. domestic flies, which were released for knowing whereabouts of pupae locations .

(2): The DNA isolation:

After the presence of large numbers of larvae inside the cage, some homogenous larvae and adults had selected to conduct DNA extraction experiments, the method of [9] has used to isolate the DNA, where used ten of larvae and adults of homogeneous insect as much as possible and must take into account the cutting parts of the insect and it larvae separately of each one, and it was well crushed or mash manually by using the peaks of high-absorbing micropipette tips which closed end edged by using Gas Bunsen Burner. phenol has been used for the purpose of extracting, The amount and purity of the isolated DNA were then estimated using the Spectrophotometer apparatus.

(3): Detecting larvae locomotion and pupae sites:

The purpose of this experiences were detecting the places of distance travelled of larvae for the purpose of entry into the pupa stage had got more than 150 homogenous larvae were in third in star with their nourishment and put inside another iron cage to prevent the cats, rodents from tampering with the samples while allowed to insects access to cage which placed in the garden of a house under the conditions of 36 ±2̊with a relative humidity of 28 ±2% in the autumn of 2017 ,then was observed the behavior of the larvae in migration and measure the distances to reach the places of pupal stage by measuring tape were recorded the distances travelled by larvae from their food to place of pupae as well as a description the hiding and pupal stages places.

RESULTS AND DISCUSSION:

(1): Isolating the DNA:

Notes from the Picture (1) described the amount of the DNA floating above the solution extracting from larvae of M. domestic flies. The analysis of samples of the DNA by using molecular methods needed to use different methods to give good and pure quantities of the DNA [10] in this study used the method of [9], it is a quick method and doesn’t take a long time as compared with other methods, the phenol compounds used for the purpose of extracting, and after that the DNA were watched as masses and agglomerated on the surface of the solution (Figure1). The results of this study showed that this method was successful to isolate a good amount of the DNA of the Iarvae, while it was inefficient to isolate the DNA of the adults where the isolated amount of adults very few, that has been attributed because of the larvae of insects in general and larvae of flies in particular, soft and that its cells continue to divide and develop and generated (growth ) until reach for stage of pupae, this led to the DNA being in replication and transcription by increasing its quantity[11]. the larvae which used in isolate the DNA were fresh and few had sold chitins modification as compared with adults ,this is consistent with what the [12]when using the larvae in molecular studies would prefer that are fresh.[13]has used the same method in which mash the Mediterranean fruit fly, Ceratitis capitate, by absorbent peak after closed it by the temperature, and thus obtained the melted DNA in solution, which is the same result which had obtained on it, it had watched the DNA with clearly in aqueous solution, despite of the different type and stage of growth the used insect. Can take advantage of DNA extraction of some insects in criminal and forensic investigations, [14], reached that DNA can be extracted from the insect of bed bugs, Cimex lectularius, which had fed on human blood through a blood meal in those insect and thus reveal the people who expected their presence in the area for the purposes of forensic investigation and criminal penalties through their sucked blood by insects which are absorbed the blood.

(2): The distances traveled by larvae of M. domestica, for pupal stages:

It seem the current experiences of the vast majority of larvae of M. domestic flies preferred immigration (crawled away) from wet food medium which reared in it to drier places for starting to pre-pupae stage, each of [15]were mentioned that the larvae of Chrysomya albiceps fly were prefer migration to driest places for start stage of pupae. When Notes figure (1) illustrates the distances which passed by the larvae of the flies ,then stop later to be it quiet preparing to pre-pupal stage it seem that many of the larvae passed a distance reach about 20 cm, the number of larvae which passed that distance reach 26 larvae, then followed by distance between 40 cm, the number of larvae in which reach 14 larvae, while the distance between 80 cm, It was the least fortunate in aspect of the number of larvae which were two larvae only, while not exceeding the number of larvae which passed a distance of 100 cm reach to five larvae, either larvae were passed a distance of more than 100 cm, where were did not exceed six larvae. The figure(1) shows some larvae did not pass any distance, the number of larvae which passed a distance below 10 cm, reach to 11 larvae. Some of larvae had preferred to remain in its place and drilling to the driest bottom, in this regard, and according to mention by[19] that larvae of M. domestic flies had dug under the piles of garbage and other debris, which were the larvae nourished on it, and formed into pupae beneath it. It was also noted the current results that most larvae preferred migration of food in the early morning hours, perhaps, the reason of that the temperatures are moderate in that time compared with the period of the afternoon are high temperatures, which may lead to drought or damage the larvae according to supported by [5].

The search for the migration of larvae and its places of pupae stage may be useful in some respects, such as its presence near the corpses bodies of some human being and secretions of animal that assist to indicate the places of their presence which helps to detect some crimes and irregularities [16] [17] mentioned that dipteran larvae which belonging to the Calliphoridae family, leave and immigrate the nutrition places in random directions preferred move toward the rising sun during rainless days.

That caused perhaps result of many of the larvae had preferred to pass a distance that does not exceed 25 cm. away from hatching eggs and nutrition habitats, that distance is important enough to be drier and far-reaching as much as possible away from the wet nutrition because they didn’t prefer walking for a long distances to avoid spending more energy from crawling away, according to confirmed this by[18]when he mentioned that in the case of the fact of nutrition and insect hatching eggs places are more dried, that make the larvae do not move a large distances for staring of pupal stage or may be not move their places or dug in the soil under the larval places then began stage of pupa.

Figure (1): The distance traveled by larvae of M.domestica for pupal stage.

(3): Detection of Pupae places of M. domestica larvae:

It appears as seem in the figure (2), that the more of areas or places which preferred by larvae M. domestica flies for forming pupae in it were under the plant residues such as leaves, small rods and others. The number of discovered larvae which turned into pupal stages places under the plant residues reached to 36 pupae out of 100 pupae had discovered then followed by the places of cracks in the walls and soil, angles of the walls and under the soil, and had been obtained on 28 larvae at the beginning of pre-pupal stages, also had found 19 pupae present under dry animal excrements and dried fruit crust. As to the least number of pupae reached 17 pupae, were picked up from the under the industrial materials non-plant such as sacks, plastics bottles, fabric and others.

The current results show that the larvae of M. domestica flies had preferred with the first rank the natural plant remains perhaps due to the abundance to fall down leaves on that place as well as some of the twigs and small rods, which have facilitated for migratory larvae chosen as places to hide and quiet beneath it, then start for pupal stages, this approximate recorded by both of[4], they indicated that the larvae of fly prefer natural plant materials piles, piles of manure and the garbage for places of pupa stages beneath it, as [19]confirmed that. As appears of the current results that the larvae after their third in star migrate look for protected, safer places to hide beneath it, this is supported by [4], they were stated that the larvae of M. domestica flies prefer immigration to dried places and hide under protected places such as garbage piles and other remains. As seemed through the result of this experience that most larvae looking for a shadow places kept away from sunlight as much as possible, that is have negative behavior toward the light of the sun, therefore it moves toward the shadow, that supported by [5], he stated that the larvae flies in general have light optical receptors to make it sensitive the light and avoid it, also he mentioned that the larvae crawl several meters, and this depends on the find anything to hide beneath it or dig inside it, if it possible that burrowed into substrate This is consistent with [20], which he found some larvae of flies burrow counted centimeters in sand, earth, dirt, or even in some fragile material to hide underneath.

Figure 2: The places where , M.domestica, larvae hid

That note after hide of the larvae under the bodies and materials become the larvae immobile and dormant , then short and limit it length to reach almost half ,and it increases diameter ,also it color begins gradual change from light white color to dark brown color when pupal stage is grow up, as show in (picture3),the highest rate or the largest number of larvae preferred hiding under the leaves of trees , plant residues and small twigs ,because of available those materials in large quantities near the places of larvae nutrition, that don’t entrusted to take efforts and trouble of moving for a long distances looking for things so as to begin pre-pupal stage beneath it, as well as the easy digging and hiding under those materials because of the light weight of that’s plants residues easy movement and lifting it hide beneath or within it scracks, as well as it good natural materials assist to fraud and camouflage far away of danger of predators because of simulation it color with the color of the larvae and pupae.[21]

Reported that blow flies species might have attracted feed generated on the corpses of bodies of animals but larvae migrated the corpses of bodies at it reach to skeletal stage the larvae after feeding on body tissues leave the body when larvae in third in star looking for another places for pupa stage[21], while species of M. domestica flies had attracted to secretions of corpses and not corpses, such as urine, excrement, vomit, blood and pus left their eggs on those areas. After the end of the larval stage or dried corpses, after that the larvae immigrate the corpses look for a convenient places for pre-pupal stages or may turn into pupa under the corpse or carrion remained of it in case of its dryness.

Figure (3):include gradual of different ages of pupae stage of M. domestica depending of the degree of color.

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Received on 12.12.2017 Modified on 15.01.2018

Research J. Pharm. and Tech. 2018; 11(3): 976-980.

DOI: 10.5958/0974-360X.2018.00182.8