Fruit Fly Behavior Lab Answers

This guide is adjusted from the Academy of Arizona Department of Biochemistry and Molecular Biophysics General Biology Programme for Science Teachers:Drosophila Melanogaster and Mendelian Genetics, by Pete Geiger.

An Introduction to Drosophila melanogaster

Drosophila melanogaster is a pocket-size, common fly found near unripe and rotted fruit. It has been in use for over a century to study genetics and behavior. Thomas Hunt Morgan was the preeminent biologist studying Drosophila early in the 1900's. He was the first to observe sexual practice-linkage and genetic recombination, which placed the small fly in the forefront of genetic research. Due to it's small size, ease of civilization and short generation time, geneticists take been using Drosophila always since.

Fruit flies are easily obtained from the wild and many biological science companies behave a variety of different mutations. In improver these companies sell any equipment needed to culture the flies. Costs are relatively depression and nearly equipment can be used twelvemonth after twelvemonth. There are a variety of laboratory exercises i could buy, although the necessity to practice so is questionable.

Why use Drosophila?

Teachers should use fruit flies for high school genetic studies for several reasons:
1. They are small and hands handled.
2. They tin can exist easily anesthetized and manipulated individually with unsophisticated equipment.
3. They are sexually dimorphic (males and females are different), making it is quite piece of cake to differentiate the sexes.
4. Virgins fruit flies are physically distinctive from mature adults, making information technology piece of cake to obtain virgin males and females for genetic crosses.
v. Flies accept a short generation time (10-12 days) and do well at room temperature.
6. The care and culture of fruit flies requires trivial equipment, is depression in price and uses footling space even for large cultures.

By using Drosophila, students will:
1. Understand Mendelian genetics and inheritance of traits
two. Draw conclusions of heredity patterns from data obtained
3. Construct traps to catch wild populations of D. melanogaster
iv. Gain an understanding of the life cycle of D. melanogaster, an insect which exhibits consummate metamorphosis
v. Construct crosses of caught and known wild- type and mutated flies
half-dozen. Learn techniques to manipulate flies, sex them, and keep concise periodical notes
7. Acquire culturing techniques to keep the flies healthy
8. Realize many science experiments cannot be conducted and concluded within one or two lab sessions

National standards covered in these lessons:
Content:
1. Organisms require a prepare of instructions for specifying traits (heredity)
ii. Hereditary information is located in genes.
iii. Combinations of traits tin can describe the characteristics of an organism.

Students goals:
1. Identify questions and concepts that guide scientific investigations
2. Pattern and conduct scientific investigations
3. Formulate and revise scientific explanations and models using logic and evidence
4. Communicate and defend a scientific argument

The genetics of Drosophila are well documented and several public-domain web sites feature the complete annotated genome. Therefore, those teachers or students wishing to run into where their mutations occur take a ready reference available.

Since Drosophila has been so widely used in genetics, in that location are many dissimilar types of mutations bachelor for buy. In addition, the attentive student may notice mutations within their ain wild-caught cultures since, due to a short generation time, mutations are relatively common compared to other animal species.

  Classification
Domain: Eukarya
Kingdom: Animalia
Phylum: Arthropoda
Course: Insecta
Order: Diptera
Family: Drosophilidae
Genus: Drosophila ("dew lover")
Species: melanogaster ("dark gut")

  Life wheel of Drosophila melanogaster
Drosophila melanogaster exhibits complete metamorphism, pregnant the life bicycle includes an egg, larval (worm-like) form, pupa and finally emergence (eclosure) equally a flying adult. This is the same every bit the well-known metamorphosis of butterflies. The larval phase has three instars, or molts.

  Twenty-four hour period 0: Female person lays eggs
Day 1: Eggs hatch
Day 2: First instar (one day in length)
Mean solar day iii: Second instar (ane day in length)
Day 5: Third and final instar (two days in length)
Day seven: Larvae brainstorm roaming stage. Pupariation (pupal germination) occurs 120 hours subsequently egg laying
Mean solar day eleven-12: Eclosion (adults emerge from the pupa case).

Females get sexually mature 8-10 hours after eclosion

• The generation time of Drosophila melanogaster varies with temperature. The higher up cycle is for a temperature of almost 22°C (72°F).  Flies raised at lower temperature (to 18°C, or 64°F) will have about twice equally long to develop.
• Females tin lay up to 100 eggs/day.
• Virgin females are able to lay eggs; however they will be sterile and few in number.

Later on the eggs hatch, pocket-size larvae should be visible in the growing medium. If your media is white, look for the small blackness surface area (the mouth hooks) at the head of the larvae. Some dried premixed media is blue to assist identify larvae however this is not a necessity and with a little patience and practice, larvae are easily seen. In add-on, equally the larvae feed they disrupt the smooth surface of the media and then by looking merely at the surface one tin tell if larvae are present. Still, information technology is ever a good thought to double cheque using a stereo microscope. Afterwards the third instar, larvae volition brainstorm to migrate up the civilisation vial in society to pupate.

Intendance, 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 whatever 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 grouping would be directly responsible for the intendance and long-term maintenance of the flies, including making large culture populations for their crosses. When straight 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 get intermixed. Losing cultures is the exception rather than the rule, and equally long every bit students re-culture their flies on a regular footing and no mass contamination occurs, flies can exist maintained for decades.

  Bottles and vials
Thomas Chase Morgan used glass milk bottles for his experiments and, indeed, any container volition do, including baby jars and assorted containers. However, for ease of culturing and transferring cultures, uniform bottles and vials are the best arroyo. Both can be purchased from a biological supply store. Bottles are used mainly for the maintenance of large populations of flies whereas civilisation vials are useful for maintaining smaller populations and are the preferred container for constructing student crosses. If at that place is a desire to maintain stock cultures for a long period of time, or to reuse bottles and vials information technology is of import completely clean and sterilize them. This is to forestall outbreaks of pests and diseases.

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

Bottles and vials can be purchased in a diversity 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 bachelor from soft cotton wool to foam plugs. This is a affair of preference and costs, nonetheless cotton fiber works fine and tin 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 await similar:

Stereo microscope

Drosophila vial

Drosophila bottles

Fly food
The commencement step in preparing culture vials is calculation food media. In that location are a variety of types of nutrient 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, and so much so that students tin can fill their own vials with media. However, it must exist 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 together dry media to the bottle or vial to about 1/five to 2/v volume. Add h2o until media appears completely moistened. Permit the vial to sit down for a few minutes, adding additional h2o if necessary until the media is completely hydrated. The surface should exist moist with a shiny appearance and there should exist no spaces in the media. If the media is non completed hydrated, production of vigorous cultures is compromised. Flies may be added minutes afterward media has been hydrated. Call up to add several grains (but non 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 ii/5th total. Keep the media out overnight to cure, keeping the vials covered with fabric to keep wild flies from laying eggs in them. The next mean solar day, add yeast and plugs. Refrigerate whatever unused media vials. Cooked media tin can be stored in a refrigerator for several weeks. Allow media to warm to room temperature earlier adding flies. Exercise not allow media to dry out out.

  Environment
The easiest way to grow flies is at room temperature. Nevertheless, 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 continue flies out of drafts and directly sunlight or estrus sources. These will quickly dry the media, necessitating frequent media changes and the potential to dehydrate the flies.

  Anesthetizing flies
The trouble with fruit flies is that they fly! Therefore a variety of methods take 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 combustible, 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 odour 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 canteen) and delivery system (vials and clamps) are necessary, increasing the costs. If resourceful, 1 tin use the CO₂ emitted from Alka-Seltzer tablets to anesthetize flies for short periods of time. Gear up upwards a large test tube with a tube and stopper system. Add together h2o in the tube, and then the Alka-Seltzer tablet. Carbon dioxide gas will be emitted.

The to the lowest degree harmful to the flies is either carbon dioxide or cooling anesthetizing. Of these ii choices, cooling is the simplest, requiring only a freezer, ice and petri dishes. In improver, information technology is the only method which will not affect fly neurology, therefore behavior studies may brainstorm after the flies have warmed up sufficiently.

  Anesthetizing flies past cooling
In order to incapacitate the flies, place the civilization 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 past using the top of a petri dish, adding crushed ice, and then placing the bottom of the petri dish on top. Adding flies to this system will keep them chilled long enough to practise each experiment. Only identify the flies back into the culture vial when finished. Flies will "wake upwards" relatively quickly one time off the ice, so go along them cold. There are no long-lasting side effects to this method, although flies left in the refrigerator as well long may non recover. Another way to keep flies chilled is adding h2o to zip-lock blazon freezer bags, place in the freezer with a petri dish nestled on the purse, and permit to freeze.

  Transferring flies from one vial to another
Flies should exist transferred every ten to xiv days. Students should maintain a backup civilization of their flies and the instructor should maintain fill-in stock cultures of all fly strains. There are two bones ways to transfer flies when forming new cultures. One requires no anesthetizing simply quick hands.
A) Identify a funnel in the oral cavity of a fresh culture vial that already has media added. In the old vial (the one with flies in it), gently tap the flies downwardly by softly tamping the vial on a soft surface, such as a mouse pad. The flies volition fall to the bottom and remain there for a few seconds (no more that!), enough time to apace take the plug off the vial, invert it into the funnel, and gently tamp, together, the two vials to forcefulness flies down into the new vial.
B) An alternative way is to put the flies in the freezer for about 8 minutes. This volition cause the flies to fall into a land of daze. After placing a funnel on the new vial, invert the vial with motionless flies into the funnel. This is not equally much fun but you won't accept any flies flying around the classroom.

  Sexing flies
It is quite piece of cake to tell males from females and with a little practice students will get 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 blackness and very distinctive merely tin only be seen nether relatively high magnification. With a little do, by looking at the abdomen students will become adept in accurately sexing flies. Sexing flies is disquisitional when making crosses, and so be certain 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 sexual practice flies and allow them to sort the flies into ii piles, male person and female person. 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 elementary matter of placing virgin females with males, it is of import to recognize the time factor involved for obtaining virgins. Females remain virgins for simply 8-ten hours after eclosure and must be collected within this time frame. Annotation: Females have the power to store sperm after a single mating, so if the female for a cross is not a virgin, you volition not know the genotype of the male person used for your cantankerous. It is strongly suggested that you obtain extra virgins in case a mistake is fabricated 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 equally virgins, they will be sterile and no larvae will be produced. Below are 3 means to obtain virgins, the 'removal method' being nigh encouraged for beginners.

  Removal method
Remove all flies 8-10 hours earlier collecting (more often than not this is done first matter in the morning). Visually inspect surface of food to ensure consummate removal of flies. Afterwards 8-10 hours (usually before yous leave piece of work) collect all females that are present. All will be virgins. Place in a fresh culture vial and wait ii-three days look for larvae. Virgin females tin can lay eggs, merely they volition be sterile. Since they are photoperiod- sensitive, females tend to eclose early on in the morning. Therefore early on collections will ensure the greatest number of virgins for experimentation. Yet, collection is possible afterward in the day.

  Visual method
Existence 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 twenty-four hours. Notation that virgin females are much larger than older females and practise not take the nighttime coloration of mature females. In improver, in the early hours after eclosure, in that location will be visible a dark greenish spot (the meconium, the remains of their last meal before pupating) on the underside of the belly.

  Temperature cycling
It is possible to maximize the number of virgins in a morn 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 ii-3 days will eliminate those 2% that are non-virgins.

  Pictures of virgin males and females:

A newly eclosed female. This is the "moisture" stage where the fly is sticky to the impact.
The wings and body have a wet appearance.

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

Comparison betwixt a mature (top) and virgin (lesser) female. This is not long after eclosure; later on 4+ hours it becomes more difficult to tell the deviation betwixt 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 even so the genitalia are distinctly different. The meconium is also plant in young virgin males as in females.

Crossing flies
In one case females are accounted virgins, add together males. When setting up crosses, a iii:1 ratio of virgin females to males is ideal. Generally, males will mate more than efficiently if they have matured iii days or longer. Be sure to select robust, healthy males; the older the flies, the lower the mating efficiency. Mating occurs rapidly and the beliefs is interesting to watch, only will not be addressed here. Females begin laying fertile eggs shortly later mating. Refer to the life cycle chart for show of F1 larvae. Remove adults in one case it has been established that enough larvae are present (typically 7-viii days afterward the cross) since you lot 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 h2o, or mineral oil is generally used. Dump anesthetized flies directly into the soapy water or mineral oil where they drown. A canteen (beaker, or spiral-capped jar) filled with ethanol or isopropanol tin can also be used as a morgue.

  Basic Drosophila Genetics Classification and Definitions

Drosophila melanogaster flies have iv chromosomes.
The genotype is written equally:

Chromosome
Chromosome or Chromosome / Chromosome

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

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

10 chromosome; chromosome Ii; chromosome III; chromosome IV

Wild-blazon is denoted as "+" or WT

Dominant mutations are written with a majuscule letter:
For case: Bar or B

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

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

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

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

Genotype – The genes that an organism possesses.

Phenotype – The observable attributes of an organism.

P1 – Parental generation.

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

F2 – The second offspring generation.

  Other great web resource:

Gerard Manning wrote a elementary introduction to Drosophila genetics.

Genetics on the Fly: A Primer on the Drosophila Model System past Karen G. Hales et al (2015).

Taking Stock of the Drosophila Research Ecosystem by David Bilder and Kenneth D. Irvine (2017).

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

Fruit Fly Behavior Lab Answers,

Source: https://depts.washington.edu/cberglab/wordpress/outreach/an-introduction-to-fruit-flies/

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