Life pupa. The pupa then develops into an imagoor

Life Cycle of Drosophila melanogasterDrosophila melanogaster, also known as the fruit fly, is a holometabolous insect sinceit has both a larval and a pupal stage prior to the adult stage (Tyler 2000). Deepa, Akshaya,and Solomon (2009) mentioned that embryonic development, following fertilization and theformation of the zygote, occurs within the egg membrane. They also stated that the eggproduces larva that eats and grows to become pupa. The pupa then develops into an imagoor adult fly (Figure 1).Figure 1. The life cycle of Drosophila melanogaster. (Weebly 2018).Temperature is an important factor that affects the duration of the life cycle. At 20°C,the average egg-larval period is 8 days while it is reduced to 5 days at 25°C (Deepa, Akshaya,and Solomon 2009). The authors indicated that the pupal stage is about 6.3 days at 20°Cwhile it is at 4.2 days at 25°C. According to them, Drosophila cultures are ideally kept at roomtemperature that does not range below 20°C or above 25°C. Furthermore, exposure totemperatures above 30°C may result in sterilization or death. On the other hand, lowtemperatures prolong the life cycle and impair the viability of the flies (Deepa, Akshaya, andSolomon 2009).Tyler (2000) mentioned that the adult Drosophila may live for more than 10 weeks andthat during this time, mating occurs. Fertilization is internal, and sperm are stored within thefemale’s body in a seminal receptacle and the paired spermathecae (Tyler 2000). Accordingto her, females reach the peak of their egg production between the fourth and seventh dayafter their emergence and around this time, they lay eggs almost continuously at a rate of 50–70 eggs per day.The eggs are about one-half mm in length (Deepa, Akshaya, and Solomon 2009; Tyler2000). They are white, oval, and slightly flattened with an ovum surrounded by an inner, verythin vitelline envelope and an outer, tough extracellular coat (Tyler 2000). This extracellularcoat is the chorion, which shows a pattern of hexagonal markings (Deepa, Akshaya, andSolomon 2009). The authors have stated that at the anterior end of the egg, a pair of smallfilaments, which are extensions of the chorion, extend from the dorsal surface and keep theegg from sinking into soft food on which it is laid. These are respiratory filaments that protrudeinto the air and serve for gas exchange (Tyler 2000).There is a small opening on the egg known as the micropyle. The penetration ofspermatozoa into the egg occurs here in the conical protrusion at the anterior end as the eggpasses through the uterus (Deepa, Akshaya, and Solomon 2009). The spermatozoa havebeen stored by the female since the mating occurred and out of the numerous sperm thatenter, only one functions in fertilization. Immediately after the entrance of the sperm, meioticdivisions are completed and the egg nucleus (female pronucleus) is formed. The authors alsosaid that the sperm nucleus and the egg nucleus would then come into position side by sideto produce the zygote nucleus, which divides to form the first two cleavage nuclei, the initialstage of development of the embryo. Eggs are laid shortly after they are penetrated by thesperm, or they may be retained in the uterus during the early stages of embryonic development(Deepa, Akshaya, and Solomon 2009).Tyler (2000) mentioned that eggs hatch in 22–24 hours at 25°C. After hatching, thelarva emerges, and it undergoes two molts, so that the larval period consists of three stages(Figure 1). The larva that emerges looks like a tiny worm and is called the first instar larva thatfeeds on the substrate that the eggs were laid in and, after another 25 hours, molts into alarger wormlike form, the second instar larva (Tyler 2000). This feeds as well and, after about24 hours, molts into the third instar larva, which is the largest of the larval forms at about 4.5mm long (Deepa, Akshaya, and Solomon 2009; Tyler 2000). Tyler said that the third instarlarva feeds, but it also starts to climb upward out of its food, so that it will be in a relativelyclean and dry area to undergo pupation. Deepa, Akshaya, and Solomon also mentioned thatthe larva is transparent and has 12 segments: the 3 head segments, 3 thoracic segments, and8 abdominal segments (Figure 2). The body wall is soft and flexible and consists of the outernoncellular cuticula and the inner cellular epidermis. A great number of sense organs are alsospread regularly over the whole body. Their fat bodies, in the form of long whitish sheets, thecoiled intestine, and the yellowish Malpighian tubules, as well as the gonads embedded in thefat body can easily be distinguished in the living larva when observed in transmitted light(Deepa, Akshaya, and Solomon 2009).The process by which the larva grows is molting and at each molt the entire cuticle ofthe insect, including many specialized cuticular structures, as well as the mouth armature andthe spiracles, is shed and has to be rebuilt again (Deepa, Akshaya, and Solomon 2009). Theysaid that the growth of the internal organs proceeds gradually and is independent of themolting process, which mainly affect the body wall and it is only the size of the cells of theinternal organs that increase while there is a constant number of cells.Figure 2. Anatomy of egg, embryo, and larva of Drosophila melanogaster. (MemorialUniversity of Newfoundland 2017).As the larva molts into a pupa after 30 hours, it becomes stationary, and in its earlystages is yellowish-white (Tyler 2000). As it develops, the pupa becomes progressively darkerand its body becomes shortened and broader. During the pupal stage, the larva ismetamorphosing into the adult fly, also called the imago (Tyler 2000). Pupation then takesplace about 12 hours after puparium formation (Deepa, Akshaya, and Solomon 2009). Bymuscular contraction the prepupa draws back from the anterior end of the puparium and evertsits head structures and larval mouth armature, which until now was attached to the anteriorend of the prepupa. The wings, halters, and legs are also everted and a typical pupa withhead, thorax, and abdomen is formed. It is seen that the pupa now lies within threemembranes: an outer membrane, the puparium: an intermediate membrane, the prepupalcuticle; and an inner membrane, the newly secreted pupal cuticle (Deepa, Akshaya, andSolomon 2009).The fly lyses most of the larval structures, with some larval organs preserved (Tyler2000). She said that the larval nervous system, for example, is not lysed, but even it undergoesmajor restructuring; the Malpighian tubules (excretory structures), fat bodies, and gonads arekept as well. Most of the adult structures, however, form anew from two sets of cells that havebeen carried as undifferentiated, mitotic cells within the larva throughout its instar stages:these are the imaginal discs (primitive cell complexes) and the histoblasts (Deepa, Akshaya,and Solomon 2009; Tyler 2000).According to Tyler (2000), the pupal stage lasts for 3–4 days, after which the adult fly,or imago, emerges from the pupal case (eclosion). Flies are fragile and light in color and theirwings are not fully expanded but they darken in a few hours and take on the normalappearance of adult flies (Deepa, Akshaya, and Solomon 2009). Adult male flies are sexuallyactive within hours of emerging while females don’t have ripe eggs until two days after eclosionsince they do not mate for about 10–12 hours after emerging. Once she has mated, she storesa considerable quantity of sperm in receptacles and fertilizes her eggs as she lays them, andthe cycle begins again (Tyler 2000).Anatomy of Drosophila melanogasterTo perform experiments on Drosophila melanogaster, one must first know its anatomy.Adult fruit flies display a typical anatomy of an insect, including compound eyes, body with 3regions—head, thorax, and abdomen—wings, and six jointed legs as depicted in Figure 3(Heard and Heard 2017). Furthermore, the ability to distinguish a male fruit fly from a femalefly is essential to do certain experiments involving the two sexes especially in the field ofgenetics.Figure 3. Body parts of a fruit fly (female). A = Head; B = Thorax; C = Abdomen; 1 =Antenna; 2 = Arista; 3 = Eye; 4 = Ocelli; 5 = Front Leg; 6 = Scutum; 7 = Middle Leg; 8 =Scutellum; 9 = Rear Body; 10 = Hind Leg; and 11 = WingThere are several characteristics that distinguish a male fruit fly from its femalecounterpart. The first difference is size. Male fruit flies are smaller in size than the female flies(Figure 4). Another characteristic that can be used to differentiate the two is the genitalia. Maleflies have dark, rounded genitalia at the tip of their abdomen while females have light, pointedgenitalia as shown in Figure 4 (Tauber Lab 2013). According to Roote and Prokop (2017), theanal plates in males are darker and more complex while in females, a pin-like extension canbe observed. Moreover, females possess a dark greenish spot in the abdomen called themeconium that indicates virginity (Figure 4, Roote and Prokop 2017).Figure 4. Ventral anatomy of a male and female fruit fly. (Camacho n.d.).Dorsally, female fruit flies display dark separated stripes at the posterior tip of theirabdomen while males have fused posterior stripes (Figure 5, Roote and Prokop 2017).Moreover, males have this structure called sex comb on the first pair of legs (Figure 5).Figure 5. Differences in male and female Drosophila (Camacho n.d.)Methods in culturing Drosophila melanogasterDrosophila melanogaster usually grows and develops in overripe and fermenting fruits.The adult fly and its larvae feed on the juices that these fruits produce. Moreover, since yeastis a main component of the fermenting fruits, Demerec and Kaufman (1996) hypothesized thatD. melanogaster also needs yeast in their diet. Thus, the fruit fly can be possibly grown in anyfermenting medium.Demerec and Kaufman (1996) and Tyler (2000) proposed three culturing media (Table1) that will provide the sugar needed for the larvae to feed and environment for the yeast togrow with a proper consistency.Table 1. Composition of culturing media for Drosophila.Banana Cornmeal Oatmeal WheatWater 47.8 mL 74.3 mL 72.7 mL 77.5 mLAgar 1.5 g 1.5 g – -Banana pulp 50.0 g – – -Molasses or Karo – 13.5 mL 11.0 mL 11.5 mLCornmeal – 10.0 g 14.0 g -Rolled oats – – 1.3 g -Cream of wheat – – – 10.3 gMethyl-p-hydroxybenzoate (10% in95% grain alcohol)0.7 mL 0.7 mL 0.7 mL 0.7 mLBanana mediumDemerec and Kaufman (1996) suggested the following method when preparing fruit flymedia as it is the easiest and fastest way of growing fruit flies. The agar is dissolved in boilingwater. The banana pulp and methyl-p-hydroxybenzoate are added afterwards and the mixtureis reheated until close to boiling. The banana pulp should be initially prepared by mashing itwith a fork or passing it through a strainer.However, Tyler (2000) proposed an easier way. Well ripened bananas are mashedand added a few drops of yeast suspension. Both methods require the media to be placed ina small glass container. If parent flies are available before hand, they are added in the mediaand the container is covered with a cotton plug. If not, the media is covered with a net, withholes large enough for fruit flies to pass through.Cornmeal mediumBennett (1961), Demerec and Kaufman (1996), and Tyler (2000) provided a simplifiedmethod of preparing the cornmeal medium for culturing Drosophila. Agar is dissolved in twothirds boiling water. Molasses is added and brought to boiling. On a separate container,cornmeal and one-thirds cold water is mixed together. These mixture is added into the boilingsolution and cooked to have consistent viscosity. Methyl-p-hydroxybenzoate is added whilestirring the mixture constantly.Cornmeal-molasses-rolled oats mediumThis medium, introduced by Demerec and Kaufman (1996) and used by Ashburnerand Roote (2007) in their study, is a modification of the cornmeal medium. Molasses and rolledoats are mixed in two-thirds boiling water. The cornmeal is mixed in cold water and added intothe boiling solution. Add methyl-p-hydroxybenzoate while stirring and cook for a few moreminutes. This medium becomes soft overtime (Demerec and Kaufman 1996). To compensate,dissolved agar with molasses and rolled oats is added to the media.Cream of wheat mediumThis medium was devised by B. Spassky of Columbia University to avoid the use ofsugar (Demerec and Kaufman 1996). Two-thirds boiling water is mixed with molasses andmethyl-p-hydroxybenzoate. The remaining cold water is mixed with Cream of Wheat andadded to the boiling solution. Stir constantly to prevent burning. After the second boil, cookuntil proper consistency is achieved. This medium tends to become soft in humid summerweather.Any of the cooked media, when ready, is added to sterilized containers. Avoid spillingon the tops or sides of bottles. In each bottle, a strip of paper toweling is placed with one endin the food for additional surface onto which the larvae can crawl and pupate. The bottles arecovered with cheesecloth and placed inside the refrigerator until the media dried out. Ifnecessary, excess moisture inside the bottles are wiped and are then plugged with bottle capswith a needle perforation.Culture set-upCulture media with methyl-p-hydroxybenzoate can be stored for a few days but mediaprepared without it should be immediately used (Demerec and Kaufman 1996). Before using,seed the food with yeast suspension, cover it in fine net with holes large enough for the fruitfly to pass through, and set it in an open area. If parent flies are available beforehand, add theparent flies into the media. When maintaining cultures, avoid overcrowding by subculturingthe fruit flies every other day.Collecting eggs and larvaeIn collecting eggs, a simple collecting chamber is prepared by using an empty culturebottle with wet toweling at the bottom and a cotton plug (Tyler 2000). The culture bottlecontaining the fruit flies is inverted into the collecting chamber and banged against a paddingto drop flies into the collecting chamber. The chamber is then quickly covered with a cottonplug.A plastic spoon containing a scored medium with yeast suspension is placed insidethe collecting chamber. To ensure that the fruit flies will not escape, a bright light is aimed atthe end of the bottle. After an hour, the spoon is collected to have new Drosophila eggs.To achieve maximum number of eggs, cultures used are five days old. During this time,the female is at its peak laying period and lays eggs every three minutes.In collecting the instar larvae, scoop the media into a petri dish. The type of instarlarvae depends on how old the culture is (Figure 1, Tyler 2000). First instar larvae can becollected 1 to 2 days after eggs are obtained. Second instar larvae are found in the media 2to 3 days after the eggs were collected while third instar larvae are found on the 3rd or 4th day.The eggs and larvae can be observed under a dissecting scope. To prevent the larvae frommoving, the larvae are placed in a drop of Ringer’s solution on a slide. The slide is placed overice in a petri dish to anesthetize the larvae.Collecting pupae and adult fliesDrosophila pupae are found on the sides of the culture bottle. This stage appears 5days after the eggs are laid. They are initially soft and white, but eventually turns brown andbrittle (Tyler 2000). Inside these pupae are fruit fly larvae that undergo metamorphosis intoadult flies.The pupae can be collected using a needle or a microknife to release them from theside of the glass. These are then examined under the dissecting scope. To remove adult flieseasily, the culture bottle is placed inside the refrigerator or kept in ice for at least 20 minutes.Demerec and Kaufman (1996) and Roote and Prokop (2017) initially suggested the use ofether or CO2 in anesthetizing, but Tyler (2000) argued that numbing the flies in ice is a saferalternative. Adult flies can be collected 8 to 10 days after eggs were laid.The adult flies can now be used for genetic studies or further culturing. The entireprocess of the study is depicted in Figure 6.