Maintaining Hawaiian Drosophila

Introduction

Most of the endemic species inhabit high elevation rain forests where temperatures rarely rise above 70°F. Also, it has been found that for many of the species exposure to temperatures much above 70°F will sterilize the males. Humidity is also important, but only in that if it is too dry, the medium will tend to dry out. We keep our Hawaiian stocks in a room that is 65-67°F (around 19°C) and greater than 50% humidity. At these conditions, we have the following timeline:

eggs      →          larvae       →     pupae     →       adults
      around 5-10 d             4-15 d          3-5 weeks

This does vary with different species somewhat.

If the stock is doing well, we tend to only make jars every other week. However, D. grimshawi, D. eurypeza and D. crucigera are always done once per week.

At the University of Hawaii (Dr. Ken Kaneshiro), they routinely use a water extract of leaves of the endemic shrub Clermontia which is not only an important oviposition stimulus for some of the species but more important, when soaked into a tissue that is placed in the vials, it is purported to contain a natural mold-inhibitor which keeps the culture medium relatively free of molds and bacteria that can affect the survivability of the adults, the eggs, and any young larvae that may hatch. Please do not omit the propionic acid from the Wheeler-Clayton food. However, at the Stock Center we only use the Clermontia leaves with D. differens.

We grow Hawaiian Drosophila on Wheeler-Clayton food. Use Wheeler-Clayton food at room temperature. This food should be papered ahead of time.

Initial Instructions

You will receive from us - one or two vials with larvae. Store vial(s) at a 30° angle in a tray until liquified food is close to the vial plug. Then, they are ready to be used for making a jar (see below).

You will receive from us - two vials with adults on a cornsyrup-based temporary food. Upon receipt, transfer all adults into new vials that contain Wheeler-Clayton food with paper. It is important that the paper in the vial, or the vials themselves, are not too wet as the flies will drown or get stuck to the side of the vial or in the paper. Do this twice per week; date and save all the vials as these should have eggs in them. Store adults and vials with eggs in the same tray as the vial with larvae and follow those instructions. Soon you will have several vials containing individuals at all different stages. Store them all paper side down and placed at a 30° angle (in the Stock Center, we lean them along a glass rod). The paper is used to keep moisture in the vial, and most importantly, to absorb any extra moisture that could drown the flies. If the paper side is not on the bottom, the flies could all die. Vials are likely to get moldy, continue to watch for larvae, these vials can still yield adults.

Making jars

Hawaiian Drosophila larvae jump into the sand and pupate on the sand. This is important in the maintenance of Hawaiian Drosophila.

Preparing jars. First get a 4 liter sized glass jar. Each jar gets 1 scoop of coarse sand (aragonite) and 1 scoop of fine sand (aragamax) - the Stock Center obtains its sand from an aquarium store, since it is pretreated. Then, using distilled (sterile) water, in a spray bottle, add a small amount (< 25 ml) to the sand in the jar. Shake the jar gently so the water gets dispersed through the sand. If this amount of water is not enough, just slowly add small amounts of water until the sand is damp throughout, not wet. YOU DO NOT WANT TO OVERWET THE SAND - YOU WILL KNOW IF YOU DID WHEN THE SAND FORMS RELATIVELY LARGE CLUMPS. You want leave space between most of the sand grains so the adults can emerge easily - if you overwet the sand it will solidify together so that you will create a barrier for the flies and they cannot reach the surface.

Once the jars are made you can start adding vials with third instar larvae. First, label the jar with the date you set up the jar and also the date(s) of the vial(s). These dates will give you an idea of how long it takes for the flies to eclose. In general, all Hawaiian flies will take about 3 weeks to eclose.

Then, spray the outside of vials with ethanol and wipe them down with a paper towel. This will remove any possible bacteria that can create mold or kill the larvae/adults.) Lay the vials on the sand at an angle and soon you will see the larvae come out. Ideally you want to place 2-4 vials in the jar, but you can use fewer at first.

Finally, put a piece of muslin (cotton) cloth (or tissue) over the jar and secure with at least 2 rubber bands. 

The sand will dry out - this is okay. If the humidity in the room is constant and about 50% you do not need to worry.

The set-up should look like this:

Transferring adults

When the adults begin to eclose in the jars, they have several weeks before they are mature. If you are unable to collect these adults as soon as they eclose, add a vial of fresh food to the jar to sustain them. Wipe this vial down with ethanol before adding it to the jar.

Collect the flies from the jar(s) into vials of Wheeler-Clayton food with paper. Label the vials with the date of eclosion. Transfer these flies once per week to new food vials and label these vials with both the eclosion date and the transfer date. Store them at an angle with the paper side down. The adults will live and lay eggs for many months. We find that the eggs tend to be laid in the moistened paper along the side of the vial.

**As an aside, if there are only a few flies in one vial or several vials, you can combine vials. It is better if you combine vials that share close dates. If you combine virgins (i.e. not sexually mature) with really old flies, you won’t be accomplishing much since no new mating will occur. You also do not want to put too many flies in one vial. As a good rule of thumb, turn the vial upside-down and tap all the flies to the plug. If you cannot see any white space, you probably have too many flies in that vial. For D. grimshawi, you do not want to put more than 12-13 in one vial as they will bite each other’s wings off. You also do not want any less than 10 because their mating success will drop.**

KEEP THE OLD VIALS. If the adults are greater than 2 weeks old, there will be larvae and the vials should be used to make jars when they are ready. Please add extra food to the vials as the larvae use it up. We find that the best thing to use is our standard cornmeal food . Store vials at an angle in a tray until the food is so liquefied (or you start to see 2nd and 3rd instar larvae), the vials are ready to be used for making jars. 

Cleaning the jars

The sand and vials are disposed of. We then clean the glass jars with soap and bleach. Rinse them thoroughly with clean water. Finally, spray the inside and outside with 70% ethanol and let them dry upside-down for a day before use.

We have adapted these from instructions kindly provided by Dr. Ken Kaneshiro.


Final note

If the goal is not to keep a stock, but to obtain some adults for a limited time, then you can maintain the Hawaiian Drosophila without the jars. If the vials remain at an angle with the paper in them, some larvae will pupate in the paper. The pupae do not eclose very well, but you do get some adults.