Information

Fruit Fly Hybrids


I have a food waste bin in which I put fruit scraps. The fruits come from all over the world, mainly Europe though.

I'm in the UK.

I assume the fruit Fly eggs are already in the fruit, in which case they originate in the same country as the fruit.

Are they all the same species? Am I creating Hybrids in my waste bin?


Probably they are not all the same species, as there are many fruit fly species in the UK and all over the world. It is also probable that you are not creating hybrids, as fruit flies have quite specific mating behaviours that change rapidly sometimes even between strains.

Anyway, you are assuming that the eggs come with the fruit, but that's hardly believable unless you already see larvae when you ate the fruit. Drosophila melanogaster has one of the fastest developments (22h since egg fertilisation, although it can vary with temperature). So, if we take for granted a week span since they were taken out of the country until you bought them (which is very optimistic) you would already see larvae in the fruit. In short, the flies that are thriving in your fruit bin are common flies from the UK.

In case you want to know, the most common flies in the UK according to this article is Drosophila subobscura and obscura, which are pretty similar in development to D. melanogaster.


Drosophila hybrid sterility

The concept of a biological species as a group of organisms capable of interbreeding to produce viable offspring dates back to at least the 18th century, although it is often associated today with Ernst Mayr. Species of the fruit-fly Drosophila are one of the most commonly used organisms in evolutionary research, and have been used to test many theories related to the evolution of species. The genus Drosophila comprises numerous species that have varying degrees of premating and postmating isolation (including hybrid sterility) between them. These species are useful for testing hypotheses of the reproductive mechanisms underlying speciation.


Fruit Flies

If you have been seeing small flies or gnats in your kitchen, they're probably fruit flies. Fruit flies can be a problem year round, but are especially common during late summer/fall because they are attracted to ripened or fermenting fruits and vegetables.

Tomatoes, melons, squash, grapes and other perishable items brought in from the garden are often the cause of an infestation developing indoors. Fruit flies are also attracted to rotting bananas, potatoes, onions and other unrefrigerated produce purchased at the grocery store. This fact sheet will explain how infestations originate and how they can be prevented in your home or place of business.

Biology and Behavior

Fruit flies are common in homes, restaurants, supermarkets and wherever else food is allowed to rot and ferment. Adults are about 1/8 inch long and usually have red eyes. The front portion of the body is tan and the rear portion is black. Fruit flies lay their eggs near the surface of fermenting foods or other moist, organic materials. Upon emerging, the tiny larvae continue to feed near the surface of the fermenting mass. This surface-feeding characteristic of the larvae is significant in that damaged or over-ripened portions of fruits and vegetables can be cut away without having to discard the remainder for fear of retaining any developing larvae. The reproductive potential of fruit flies is enormous given the opportunity, they will lay about 500 eggs. The entire lifecycle from egg to adult can be completed in about a week.

Fruit flies are especially attracted to ripened fruits and vegetables in the kitchen. But they also will breed in drains, garbage disposals, empty bottles and cans, trash containers, mops and cleaning rags. All that is needed for development is a moist film of fermenting material. Infestations can originate from over-ripened fruits or vegetables that were previously infested and brought into the home. The adults can also fly in from outside through inadequately screened windows and doors.

Fruit flies are primarily nuisance pests. However, they also have the potential to contaminate food with bacteria and other disease-producing organisms.

Prevention

The best way to avoid problems with fruit flies is to eliminate sources of attraction. Produce which has ripened should be eaten, discarded or refrigerated. Cracked or damaged portions of fruits and vegetables should be cut away and discarded in the event that eggs or larvae are present in the wounded area. A single rotting potato or onion forgotten at the back of a closet, or fruit juice spillage under a refrigerator can breed thousands of fruit flies. So can a recycling bin stored in the basement which is never emptied or cleaned.

People who can their own fruits and vegetables, or make wine, cider or beer should ensure that the containers are well sealed otherwise, fruit flies will lay their eggs under the lid and the tiny larvae will enter the container upon hatching. Windows and doors should be equipped with tight-fitting (16 mesh) screens to help prevent adult fruit flies from entering from outdoors.

Eradication

Once a structure is infested with fruit flies, all potential breeding areas must be located and eliminated. Unless the breeding sites are removed or cleaned, the problem will continue no matter how often insecticides are applied to control the adults. Finding the source(s) of attraction and breeding can be very challenging and often will require much thought and persistence. Potential breeding sites which are inaccessible (e.g., garbage disposals and drains) can be inspected by taping a clear plastic food storage bag over the opening overnight. If flies are breeding in these areas, the adults will emerge and be caught in the bag.

After the source of attraction and breeding is eliminated, a pyrethrum-based, aerosol insecticide may be used to kill any remaining adult flies in the area.

A better approach, however, is to construct a trap by placing a paper funnel (rolled from a sheet of notebook paper) into a jar which is then baited with a few ounces of cider vinegar. Place the jar trap(s) wherever fruit flies are seen. This simple but effective trap will soon catch any remaining adult flies which can then be killed or released outdoors.

CAUTION! Pesticide recommendations in this publication are registered for use in Kentucky, USA ONLY! The use of some products may not be legal in your state or country. Please check with your local county agent or regulatory official before using any pesticide mentioned in this publication.

Of course, ALWAYS READ AND FOLLOW LABEL DIRECTIONS FOR SAFE USE OF ANY PESTICIDE!


Geneticists discover genes that make fruit fly hybrids sterile

While hybrids -- the result of the mating of two different species -- may offer interesting and beneficial traits, they are usually sterile or unable to survive. For example, a mule, the result of the mating of a horse and a donkey, is sterile.

Now, Cornell researchers have made the first identification of a pair of genes in any species that are responsible for problems unique to hybrids. Specifically, the researchers have found two genes from two fruit fly species (Drosophila melanogaster and D. simulans) that interfere with each other, thereby preventing the production of male offspring.

The finding may eventually shed light on what causes lethality or sterility in hybrids in general and, in a larger sense, offers clues to how species evolve from common ancestors.

The research, published in the Nov. 24 issue of Science, focuses on a rarely occurring mutation in a D. melanogaster gene called "Hmr" (Hybrid male rescue) and a similar mutation in a D. simulans gene called "Lhr" (Lethal hybrid rescue) that make these genes nonfunctional. When either of these genes is "turned off" and then crossed with the other fruit fly species, the males survive.

"We have found the first example of two genes that interact to cause lethality in a species hybrid," said the paper's senior author, Daniel Barbash, assistant professor in Cornell's Department of Molecular Biology and Genetics.

The finding supports the Dobzhansky-Muller model, a theory from the 1930s that suggests hybrid incompatibilities (such as death or sterility) are caused by genes that have evolved from a common ancestor but diverged in each of the species. More specifically, in the common ancestor, these genes may have worked perfectly well together. But, as each gene evolved in its own species, it began to code for proteins that no longer work in the other species.

In this case, when genes from each species were compared with each other, the Hmr gene in D. melanogaster and the Lhr gene in D. simulans each evolved much faster than most other genes and diverged due to natural selection, a genetic change due to a pressure that benefits the survival of a species. The researchers would like to learn what these genes normally do within their species in order to understand why they are evolving so fast.

The Dobzhansky-Muller model also proposes that these evolved genes depend on each other to cause hybrid incompatibilities.

However, when Barbash and his colleagues cloned each gene and inserted an Lhr gene from D. simulans into D. melanogaster, the two genes did not interfere with each other in the engineered D. melanogaster strain even though the Lhr and Hmr genes interfere with each other in hybrids.

"This tells us there must be other things involved in the hybrid" that impacts the incompatible pairing of these genes, said Barbash. In future work the researchers hope to determine whether the hybrids die because of additional genes like Hmr and Lhr, or because of more subtle differences between the chromosomes of these two species.


Geneticists discover genes that make fruit fly hybrids sterile

While hybrids -- the result of the mating of two different species -- may offer interesting and beneficial traits, they are usually sterile or unable to survive. For example, a mule, the result of the mating of a horse and a donkey, is sterile.

Now, Cornell researchers have made the first identification of a pair of genes in any species that are responsible for problems unique to hybrids. Specifically, the researchers have found two genes from two fruit fly species (Drosophila melanogaster and D. simulans) that interfere with each other, thereby preventing the production of male offspring.

The finding may eventually shed light on what causes lethality or sterility in hybrids in general and, in a larger sense, offers clues to how species evolve from common ancestors.

The research, published in the Nov. 24 issue of Science, focuses on a rarely occurring mutation in a D. melanogaster gene called "Hmr" (Hybrid male rescue) and a similar mutation in a D. simulans gene called "Lhr" (Lethal hybrid rescue) that make these genes nonfunctional. When either of these genes is "turned off" and then crossed with the other fruit fly species, the males survive.

"We have found the first example of two genes that interact to cause lethality in a species hybrid," said the paper's senior author, Daniel Barbash, assistant professor in Cornell's Department of Molecular Biology and Genetics.

The finding supports the Dobzhansky-Muller model, a theory from the 1930s that suggests hybrid incompatibilities (such as death or sterility) are caused by genes that have evolved from a common ancestor but diverged in each of the species. More specifically, in the common ancestor, these genes may have worked perfectly well together. But, as each gene evolved in its own species, it began to code for proteins that no longer work in the other species.

In this case, when genes from each species were compared with each other, the Hmr gene in D. melanogaster and the Lhr gene in D. simulans each evolved much faster than most other genes and diverged due to natural selection, a genetic change due to a pressure that benefits the survival of a species. The researchers would like to learn what these genes normally do within their species in order to understand why they are evolving so fast.

The Dobzhansky-Muller model also proposes that these evolved genes depend on each other to cause hybrid incompatibilities.

However, when Barbash and his colleagues cloned each gene and inserted an Lhr gene from D. simulans into D. melanogaster, the two genes did not interfere with each other in the engineered D. melanogaster strain even though the Lhr and Hmr genes interfere with each other in hybrids.

"This tells us there must be other things involved in the hybrid" that impacts the incompatible pairing of these genes, said Barbash. In future work the researchers hope to determine whether the hybrids die because of additional genes like Hmr and Lhr, or because of more subtle differences between the chromosomes of these two species.

The study was funded by the National Institutes of Health.

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.


Insect Declines in the Anthropocene

David L. Wagner
Vol. 65, 2020

Abstract

Insect declines are being reported worldwide for flying, ground, and aquatic lineages. Most reports come from western and northern Europe, where the insect fauna is well-studied and there are considerable demographic data for many taxonomically disparate . Read More

Figure 1: Location of 73 insect decline reports by taxon or group, adapted from Sánchez-Bayo & Wyckhuys (156). Each square represents a single study, with the base of each stacked bar positioned over .

Figure 2: Population trends for insects tracked by the International Union for Conservation of Nature (IUCN) and UK insects from Dirzo et al. (34). (a) Trend data for IUCN-listed Coleoptera (Col), Hym.

Figure 3: Reversal of fortunes. An important aspect of recent decline reports is evidence of steep population declines in formerly abundant species. (a) The Rocky Mountain locust (Melanoplus spretus)—.


3 Ways to Get Rid of Fruit Flies

Getting rid of fruit flies goes hand in hand with preventing them from multiplying. If you kill as many adult flies as possible while eliminating their food sources and breeding grounds, you're on your way to winning the battle.

Eliminate Food Sources

The first step to getting rid of fruit flies is identifying all potential sources that support them, then doing away with (or putting away) those sources. In addition to fruit and vegetables, these tiny flies like sweet stuff (like spilled juice or jelly), fermented stuff (open beer cans or wine bottles), and rotten stuff (slime inside garbage disposers and drains). For the same reasons, they also like garbage, recycling cans, and compost containers. Most fruits can be stored in the fridge (where flies can't get to them). All other food sources for fruit flies should be cleaned up or taken out.

The Spruce / Cristina Tudor

Try Some Traps

DIY fruit fly traps are all over the internet, and they're all inexpensive and easy to make. Do they work? Sometimes. But they're so simple that it can't hurt to try. Traps range from a bowl full of vinegar to contraptions made from plastic soda bottles reminiscent of grade-school science projects. The idea is to set out some traps on the kitchen counter, wherever fruit flies hang out. The flies are attracted to the liquid in the trap, then they get trapped or they drown. You'll catch adults only the young ones are eating and growing right on the food source, but catching a single adult can potentially prevent hundreds of new flies. You can also buy sticky fly traps designed for fruit flies. Many of these include a bait on the sticky surface to lure the flies.

The Spruce / Cristina Tudor

Spray Them (If You Want)

Fruit flies usually are not enough of a nuisance to warrant spraying toxic insecticide around your kitchen, but this can be a tempting option if they're driving you crazy. As with traps, it works only on the adults. Don't spray your food in an effort to kill young flies because you'll just ruin the food. While commercial sprays such as permethrin are effective on fruit flies, you can also spray them with 91-percent isopropyl alcohol (rubbing alcohol), using a fine-mist sprayer. Alcohol is a great sanitizer, and it doesn't harm most surfaces, but it can discolor some materials.

The Spruce / Cristina Tudor


Researchers use fruit flies to unlock mysteries of human diabetes

Researchers have developed a technique to measure insulin levels in fruit flies, promoting the uses of this insect for diabetes research.

Seung Kim is the senior author of a paper that describes a technique for measuring insulin levels in fruit flies, giving researchers a powerful new way to study diabetes.

For the first time, the tiny fruit fly can be used to study how mutations associated with the development of diabetes affect the production and secretion of the vital hormone insulin.

The advance is due to a new technique devised by researchers at the Stanford University School of Medicine that allows scientists to measure insulin levels in the insects with extremely high sensitivity and reproducibility.

The experimental model is likely to transform the field of diabetes research by bringing the staggering power of fruit fly genetics, honed over 100 years of research, to bear on the devastating condition that affects millions of Americans. Until now, scientists wishing to study the effect of specific mutations on insulin had to rely on the laborious, lengthy and expensive genetic engineering of laboratory mice or other mammals.

In contrast, tiny, short-lived fruit flies can be bred in dizzying combinations by the tens of thousands in just days or weeks in small flasks on a laboratory bench.

Seung Kim, MD, PhD, professor of developmental biology, is the senior author of the paper describing the research. Research associate Sangbin Park, PhD, is the lead author of the paper, published Aug. 7 in PLOS Genetics.

Developed by Park, the new technique uses a chemical tag to label an insulin-like peptide called Ilp2 in fruit flies. The tag allows researchers to use an antibody-based assay to measure insulin concentrations in the insect’s blood and cells at the picomolar level — the level at which insulin concentrations are measured in humans.

“I normally avoid the term, but I think Dr. Park’s new technique is a true breakthrough,” said Kim, who is also a Howard Hughes Medical Institute investigator. “Only in selected mammals can researchers measure insulin with this degree of sensitivity.”

The power of a tiny model system

Insulin is an ancient molecule used by nearly all animals to regulate metabolism, growth and development. Diabetes in humans occurs when insulin-making cells in the pancreas fail to produce the hormone or when other cells in the body grow resistant to its effects. In 2002, Kim, his lab team and fellow Stanford researchers discovered that fruit flies develop a diabetes-like condition when their insulin-producing cells are destroyed.

“Studies of diabetes in fruit flies represent a relatively new area of investigation,” said Carl Thummel, PhD, professor of human genetics at the University of Utah School of Medicine. Thummel uses the insect to study energy metabolism and metabolic disorders.

“Needless to say, fruit flies are very small, and only tiny amounts of blood can be extracted from their bodies,” he said. “Our inability to measure the amounts of circulating insulin has been a major drawback in the field. The technique developed by Dr. Kim’s group will allow researchers to rapidly test the effect of diabetes risk factors, and establishes fruit flies as an effective tool for studies of diabetes.”

Using the technique, the researchers were able to quickly identify what a mutation associated with type-2 diabetes in humans actually does: It regulates insulin secretion, but not production.

Understanding the effect of each mutation

Parsing the effect of each mutation on the way the body produces, secretes and responds (or not) to insulin is critical to further understand the disease and to devise new therapeutic approaches. “I was stunned that this technique worked so well to identify the effect of specific mutations,” said Park. “Many of the genes we studied seem to have similar functions in governing insulin production or secretion in flies and in humans.”

Previous efforts to tag Ilp2 have been hampered by the fact that the protein undergoes a complex series of modifications and folding events on its way to becoming the active form of the molecule. Tags that disrupt this process can cause inappropriate expression of the molecule or render it inactive, interfering with the very metabolic pathway researchers want to study.

Park capitalized on the knowledge that overexpression of the active form of the Ilp2 protein is lethal. He then randomly inserted chemical tags along the length of the molecule to create a panel of molecules tagged in many different places. Testing them individually, he looked for those that were still able to kill the flies — indicating that the molecule’s activity had not been compromised. Eventually he found two locations on Ilp2 that were ideal. He could then use antibodies that recognized the tags to quantify levels of Ilp2 with the antibody-based assay.

“Once you know that the modifications, or tags, don’t affect the expression or activity of the molecule, you have a lot more power to interpret your experiments,” said Kim. “You can begin to track the insulin assembly line, from the transcription of RNA from the gene, to the production of the protein, to the storing and eventual secretion of the protein in response to metabolic signals. You have the opportunity to figure out the mechanisms controlling each of those steps in detail.”

In flies, Ilp2 is produced and secreted by specialized neurons in the brain. This makes it relatively easy to compare levels of circulating Ilp2 with the amount of mature but unsecreted Ilp2: simply compare the amount of Ilp2 in the insects’ bodies to the amount in their brains.

Park found that the amount of secreted Ilp2 increased from about 0.1 percent to about 0.35 percent of the total available during the first three days of a fruit fly’s life. Furthermore, like in humans, circulating Ilp2 concentrations were relatively low in fasting flies, but peaked and then declined after a subsequent meal. Finally he showed that, in flies with only one working copy of the insulin receptor gene (they normally have two, as do humans), insulin secretion was increased in an apparent attempt to compensate for the deficiency — mirroring the development of insulin resistance in humans and mice.

Park and his colleagues then turned their attention to mutations associated with type-2 diabetes in genome-wide studies in humans. These studies don’t reveal how a specific mutation might work to affect development of a disease they show only that people with the condition are more likely than those without it to have certain mutations in their genome. Hundreds of candidate-susceptibility genes have been identified in this way.

Tip of the iceberg

The researchers found that blocking the expression of a fly version of a human protein called GLIS3, known to affect insulin production in mammals, and linked both to type-2 and type-1 diabetes in humans, also affected the production of Ilp2 in flies. A mutation in another protein, BCL11A, was known to be associated with the development of the disease in humans, but its mechanism of action was unclear. Park and his colleagues found that blocking the expression of the fly version of BCL11A did not affect the flies’ ability to make Ilp2, but caused it to secrete abnormally high levels of Ilp2 into the bloodstream.

The researchers emphasize that these findings are just the tip of the iceberg. Many more mutations can be studied alone and in combination under a myriad of experimental conditions. A single fruit fly can lay several hundred eggs during its approximately 40-day life span eggs develop into adults in only 10 days. They plan to continue to use the fruit fly system to complement and inform their ongoing studies in mammals and humans.

“We’re really taking advantage of a century of work done by generations of other researchers,” said Kim. “Historically the fly has been used to understand developmental biology by looking at its genes and its cells and observing how they change over time. Now we’ve shown we can accurately and precisely measure levels of a crucial hormone in these insects, and use that to identify new targets for diabetes investigation in mice and humans.”

Other authors are Stanford graduate student Ronald Alfa and research associate Lutz Kockel, and high-school students Grace Kim and Sydni Topper.


Interactions between Drosophila and its natural yeast symbionts-Is Saccharomyces cerevisiae a good model for studying the fly-yeast relationship?

Yeasts play an important role in the biology of the fruit fly, Drosophila melanogaster. In addition to being a valuable source of nutrition, yeasts affect D. melanogaster behavior and interact with the host immune system. Most experiments investigating the role of yeasts in D. melanogaster biology use the baker's yeast, Saccharomyces cerevisiae. However, S. cerevisiae is rarely found with natural populations of D. melanogaster or other Drosophila species. Moreover, the strain of S. cerevisiae used most often in D. melanogaster experiments is a commercially and industrially important strain that, to the best of our knowledge, was not isolated from flies. Since disrupting natural host-microbe interactions can have profound effects on host biology, the results from D. melanogaster-S. cerevisiae laboratory experiments may not be fully representative of host-microbe interactions in nature. In this study, we explore the D. melanogaster-yeast relationship using five different strains of yeast that were isolated from wild Drosophila populations. Ingested live yeasts have variable persistence in the D. melanogaster gastrointestinal tract. For example, Hanseniaspora occidentalis persists relative to S. cerevisiae, while Brettanomyces naardenensis is removed. Despite these differences in persistence relative to S. cerevisiae, we find that all yeasts decrease in total abundance over time. Reactive oxygen species (ROS) are an important component of the D. melanogaster anti-microbial response and can inhibit S. cerevisiae growth in the intestine. To determine if sensitivity to ROS explains the differences in yeast persistence, we measured yeast growth in the presence and absence of hydrogen peroxide. We find that B. naardenesis is completely inhibited by hydrogen peroxide, while H. occidentalis is not, which is consistent with yeast sensitivity to ROS affecting persistence within the D. melanogaster gastrointestinal tract. We also compared the feeding preference of D. melanogaster when given the choice between a naturally associated yeast and S. cerevisiae. We do not find a correlation between preferred yeasts and those that persist in the intestine. Notably, in no instances is S. cerevisiae preferred over the naturally associated strains. Overall, our results show that D. melanogaster-yeast interactions are more complex than might be revealed in experiments that use only S. cerevisiae. We propose that future research utilize other yeasts, and especially those that are naturally associated with Drosophila, to more fully understand the role of yeasts in Drosophila biology. Since the genetic basis of host-microbe interactions is shared across taxa and since many of these genes are initially discovered in D. melanogaster, a more realistic fly-yeast model system will benefit our understanding of host-microbe interactions throughout the animal kingdom.

Keywords: Baker’s yeast Drosophila melanogaster Host-microbe interactions Microbiome Microbiota Saccharomyces cerevisiae Symbiosis Yeast.

Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1. Persistence of yeasts in the…

Figure 1. Persistence of yeasts in the D. melanogaster intestine relative to Saccharomyces cerevisiae .


Care, Maintenance and Manipulation of Drosophila

Introduction
In order to incorporate fruit flies in the classroom, it will be necessary to maintain cultures of flies for manipulation in crosses and as a backup for any mishaps which may occur. Culturing is very easy and it is recommended to have students maintain their own cultures of flies. In that way, each student or group would be directly responsible for the care and long-term maintenance of the flies, including making large culture populations for their crosses. When directly involved, students gain proficiency and a greater understanding of the flies requirements and behavior. The teacher should remain as coach, not lecturer, assisting students in techniques. The instructor needs to maintain stock cultures of all strains and mutants used by students in case the aforementioned unforeseeable incident occurs and student cultures die out or become intermixed. Losing cultures is the exception rather than the rule, and as long as students re-culture their flies on a regular basis and no mass contamination occurs, flies can be maintained for decades.


Bottles and vials
Thomas Hunt Morgan used glass milk bottles for his experiments and, indeed, any container will do, including baby jars and assorted containers. However, for ease of culturing and transferring cultures, uniform bottles and vials are the best approach. Both can be purchased from a biological supply store. Bottles are used mainly for the maintenance of large populations of flies whereas culture vials are useful for maintaining smaller populations and are the preferred container for constructing student crosses. If there is a desire to maintain stock cultures for a long period of time, or to reuse bottles and vials it is important completely clean and sterilize them. This is to prevent outbreaks of pests and diseases.

To clean bottle and vials, first freeze them to kill any flies in them. Remove the food, wash well, then sterilize by autoclaving (for 20 minutes at 121°C and 15 psi if containers are plastic, be sure they can be autoclaved) or washing in a 10% chlorine bleach solution.

Bottles and vials can be purchased in a variety of sizes and materials. Glass is effective, however if dropped a student could lose 2 weeks of data in a single spill. Autoclaved (sterile) plastic vials are available and are preferable for student use. Vial sizes range from 96 mm by 25 mm to larger sizes, however the smaller size is recommended for making crosses and maintaining small cultures. There are a variety of plugs available from soft cotton to foam plugs. This is a matter of preference and costs, however cotton works fine and can be bought at a local drug store in a pinch.


Where to buy supplies:
Carolina Biological Supply Company
FlyStuff.com, A division of Genesee Scientific


What they look like:

Stereo microscope Drosophila vial Drosophila bottles


Fly food
The first step in preparing culture vials is adding food media. There are a variety of types of food available for the flies some require cooking and others are bought already prepared and dehydrated. The latter can be purchased from a biological supply company. This is, of course, much quicker and easier than preparing cooked media, so much so that students can fill their own vials with media. However, it must be completely rehydrated for best results, since this is the only water source for adults and larvae. Therefore, follow the suggestions below to ensure a completely hydrated media:

Dehydrated media
Add dry media to the bottle or vial to about 1/5 to 2/5 volume. Add water until media appears completely moistened. Allow the vial to sit for a few minutes, adding additional water if necessary until the media is completely hydrated. The surface should be moist with a shiny appearance and there should be no spaces in the media. If the media is not completed hydrated, production of vigorous cultures is compromised. Flies may be added minutes after media has been hydrated. Remember to add several grains (but not more) of yeast to the media surface before adding flies.

Cooked media
When dispensing cooked media, it should fill the culture vial, bottle or vial 1/5th to 2/5th full. Keep the media out overnight to cure, keeping the vials covered with cloth to keep wild flies from laying eggs in them. The next day, add yeast and plugs. Refrigerate any unused media vials. Cooked media can be stored in a refrigerator for several weeks. Allow media to warm to room temperature before adding flies. Do not allow media to dry out.


Environment
The easiest way to grow flies is at room temperature. However, the optimum rearing condition is a temperature of 25°C and 60% humidity. In these conditions generation time is shorter (9-10 days from egg to adult). Unless equipment is readily available this is unnecessary for successful rearing and crossing of flies. It is preferable to keep flies out of drafts and direct sunlight or heat sources. These will rapidly dry the media, necessitating frequent media changes and the potential to dehydrate the flies.


Anesthetizing flies
The problem with fruit flies is that they fly! Therefore a variety of methods have been developed to anesthetize flies. Include are ether, commercial brands such as Flynap, carbon dioxide, and cooling. Each has its strengths and weaknesses. Ether is flammable, has a strong odor and will kill flies if they are over-etherized (and can anesthetize younger students!). Flynap, from Carolina Biological, is messy and has an odor that some find offensive. Each of these, however, requires low-cost equipment which can be easily purchased. Carbon dioxide works very well, keeping flies immobile for long periods of time with no side effects, however CO2 mats (blocks) are expensive and a CO2 source (usually a bottle) and delivery system (vials and clamps) are necessary, increasing the costs. If resourceful, one can use the CO2 emitted from Alka-Seltzer tablets to anesthetize flies for short periods of time. Set up a large test tube with a tube and stopper system. Add water in the tube, then the Alka-Seltzer tablet. Carbon dioxide gas will be emitted.

The least harmful to the flies is either carbon dioxide or cooling anesthetizing. Of these two choices, cooling is the simplest, requiring only a freezer, ice and petri dishes. In addition, it is the only method which will not affect fly neurology, therefore behavior studies may begin after the flies have warmed up sufficiently.


Anesthetizing flies by cooling
In order to incapacitate the flies, place the culture vial in the freezer until the flies are not moving, generally 8-12 minutes. Dump the flies onto a chilled surface. This can be constructed by using the top of a petri dish, adding crushed ice, then placing the bottom of the petri dish on top. Adding flies to this system will keep them chilled long enough to do each experiment. Simply place the flies back into the culture vial when finished. Flies will “wake up” relatively quickly once off the ice, so keep them cold. There are no long-lasting side effects to this method, although flies left in the refrigerator too long may not recover. Another way to keep flies chilled is adding water to zip-lock type freezer bags, place in the freezer with a petri dish nestled on the bag, and allow to freeze.


Transferring flies from one vial to another
Flies should be transferred every 10 to 14 days. Students should maintain a backup culture of their flies and the instructor should maintain backup stock cultures of all fly strains. There are two basic ways to transfer flies when forming new cultures. One requires no anesthetizing but quick hands.
A) Place a funnel in the mouth of a fresh culture vial that already has media added. In the old vial (the one with flies in it), gently tap the flies down by softly tamping the vial on a soft surface, such as a mouse pad. The flies will fall to the bottom and remain there for a few seconds (no more than that!), enough time to quickly take the plug off the vial, invert it into the funnel, and gently tamp, together, the two vials to force flies down into the new vial.
B) An alternative way is to put the flies in the freezer for about 8 minutes. This will cause the flies to fall into a state of stupor. After placing a funnel on the new vial, invert the vial with motionless flies into the funnel. This is not as much fun but you won’t have any flies flying around the classroom.


Sexing flies
It is quite easy to tell males from females and with a little practice students will become confident of their ability to do so. Notice that males are generally smaller and have a darker and more rounded abdomen. The coloration of the abdomen is the easiest to recognize. In addition, males have tarsal sex combs on their first pair of legs. These are black and very distinctive but can only be seen under relatively high magnification. With a little practice, by looking at the abdomen students will become proficient in accurately sexing flies. Sexing flies is critical when making crosses, so be sure student are confident in identifying the difference between the sexes. In order for students to feel comfortable sexing flies, give or have them obtain 25 or more mixed sex flies and allow them to sort the flies into two piles, male and female. Other students in the group and the instructor should verify the sorting. Each member of the group should be able to sex flies.


Pictures of males and females

Ventral view of a male (top) and female (bottom).

Lateral view of a male (top) and female (bottom).

Note the darker abdomen and more rounded appearance of the male. Females also tend to be larger.


Collecting virgin females
While it’s a simple matter of placing virgin females with males, it is important to recognize the time factor involved for obtaining virgins. Females remain virgins for only 8-10 hours after eclosure and must be collected within this time frame. NOTE: Females have the ability to store sperm after a single mating, so if the female for a cross is not a virgin, you will not know the genotype of the male used for your cross. It is strongly suggested that you obtain extra virgins in case a mistake is made in identification or the fly dies before mating and egg lying can occur. In a strong culture, multiple virgin females should be easily obtained. Although females are able to lay eggs as virgins, they will be sterile and no larvae will be produced. Below are three ways to obtain virgins, the ‘removal method’ being most encouraged for beginners.


Removal method
Remove all flies 8-10 hours before collecting (generally this is done first thing in the morning). Visually inspect surface of food to ensure complete removal of flies. After 8-10 hours (usually before you leave work) collect all females that are present. All will be virgins. Place in a fresh culture vial and wait 2-3 days look for larvae. Virgin females can lay eggs, but they will be sterile. Since they are photoperiod- sensitive, females tend to eclose early in the morning. Therefore early collections will ensure the greatest number of virgins for experimentation. However, collection is possible later in the day.


Visual method
Being able to recognize virgin females removes the necessity of emptying culture vials on a timely basis and allows students to collect their own without the necessity of coming to class at odd times of the day. Note that virgin females are much larger than older females and do not have the dark coloration of mature females. In addition, in the early hours after eclosure, there will be visible a dark greenish spot (the meconium, the remains of their last meal before pupating) on the underside of the abdomen.


Temperature cycling
It is possible to maximize the number of virgins in a morning collection by using temperature cycling. When cultures are maintained at a temperature of 18°C, development is slowed so females will not mate until 16 hours after enclosure. By removing flies in the afternoon/evening and placing the vials in an 18°C incubator, 98% of flies obtained in the morning will be virgins. Placing virgins in their own vials for 2-3 days will eliminate those 2% that are non-virgins.


Pictures of virgin males and females:

A newly eclosed female. This is the “wet” stage where the fly is sticky to the touch.
The wings and body have a wet appearance.

Virgin female showing the meconium (arrow).
The meconium is a dark green area and is the remains of larval food

Comparison between a mature (top) and virgin (bottom) female. This is not long after eclosure after 4+ hours it becomes more difficult to tell the difference between the two.
Note the meconium on the virgin female.

Comparison between a mature (top) and virgin (bottom) male. The coloration is similar to virgin females however the genitalia are distinctly different. The meconium is also found in young virgin males as in females.

Crossing flies
Once females are deemed virgins, add males. When setting up crosses, a 3:1 ratio of virgin females to males is ideal. Generally, males will mate more efficiently if they have matured 3 days or longer. Be sure to select robust, healthy males the older the flies, the lower the mating efficiency. Mating occurs quickly and the behavior is interesting to watch, but will not be addressed here. Females begin laying fertile eggs soon after mating. Refer to the life cycle chart for evidence of F1 larvae. Remove adults once it has been established that enough larvae are present (typically 7-8 days after the cross) since you may not be able to distinguish parents from the F1 generation.


Killing Flies: The Morgue
This is an unfortunate necessity when using flies. A bottle or beaker with soapy water, or mineral oil is generally used. Dump anesthetized flies directly into the soapy water or mineral oil where they drown. A bottle (beaker, or screw-capped jar) filled with ethanol or isopropanol can also be used as a morgue.


Basic Drosophila Genetics Nomenclature and Definitions

Drosophila melanogaster flies have 4 chromosomes.
The genotype is written as:

Chromosome
Chromosome or Chromosome / Chromosome

This common nomenclature shows one chromosome on top and its homologue on the bottom, as the chromosomes would appear during meiosis when contributing gametes.

When writing the genotype, in general, chromosomes are separated with a semicolon.

X chromosome chromosome II chromosome III chromosome IV

Wild-type is denoted as “+” or WT

Dominant mutations are written with a capital letter:
For example: Bar or B

Recessive mutations are written with a lower case letter:
For example: white or w

Mutations are alleles (alternative forms of a gene occupying a given locus on a chromosome) that are inherited with chromosomes.

Homozygote – An individual with the same allele at corresponding loci on the homologous chromosomes.

Heterozygote – An individual with different alleles at corresponding loci on the homologous chromosomes.

Genotype – The genes that an organism possesses.

Phenotype – The observable attributes of an organism.

F1 – Filial generation, or offspring generation. F1 is the first offspring generation.

F2 – The second offspring generation.


Other great web resources:

FlyBase is an encyclopedic resource for Drosophila researchers, with detailed information on fly stocks, genes, mutants, researchers, publications and much more.