7.Pan sorting (after sieving):

a. Adults- Count or sort the beetles collected in the aluminum pan by brushing adults into a petri dish with a small to medium sized brush. If your sample has a very large number of adults in it, flying beetles can be a problem. (Beetles seem to get more excited and want to fly away when crowded.) You can minimize the problem by first putting all the collected beetles in one or more petri dishes and place lids on the dishes. Then return smaller portions of beetles to the aluminum pan for sorting a bit at a time.

b. Pupae- If collecting pupae from a jar with a spoon, you can exclude many adults by tilting the jar to one side. Adults will move to the low side, and you can scoop from the center (Be sure to "sterilize" the spoon first by wiping off and rapping it against the table top several times on both sides!). Sieve, then brush adults and larvae into one petri dish, and brush pupae into another dish. (The collected adults and larvae should be discarded unless the population of that stock is so low that you need to use every beetle to insure survival of the stock.)

Note: Sorting adults, pupae, or larvae with a brush is easier if accumulations of exuvia (cast-off skins) are first removed. One method to remove them is by gently blowing them out of the pan, using a side-to-side and near-to-far sweeping motion with your breath, blowing them into a waste basket. It usually takes 3 to four "sweepings" to get most of the exuvia out. (Be careful to blow gently enough that only exuvia, and some dead adults are blown out --not the live adults, pupae and larvae. Dead beetles and exuvia are lighter than live ones and careful blowing helps to separate them.)

Another way of separating pupae from adults and larvae is to sift the whole jar, place the adults, larvae and pupae (the siftings), into a petri dish or other clean container, then work with small amounts of the siftings. For each lot, blow off the skins, then shake down the adults and pupae, leaving the larvae. Pour the adults and pupae onto a petri dish lid in a covered sieve receiving pan, and let the adults run off, leaving mostly pupae.

Exuvia can also be removed by vacuuming the siftings (from the bottomof the sieve) before placing in the aluminum sorting pan.

8.Use of topping- Topping (coarsely ground wheat) is used to give beetles traction on the flour so they can right themselves if they fall onto their backs (while many beetles in a container can help each other get up, a lone beetle can get stranded on its back and starve to death!). Use topping if:

a. Population density is low due to disease or mutation

b. Adults have impaired ability to right themselves due to a mutation affecting leg size or shape. For instance, it is wise to use topping with stocks of Dch3* since they can't get around as well as beetles with normal sized and shaped front legs, and since they have lower fertility than other strains.

9.Subculturing schedule- If using a 30° C. incubator temperature, subculture heavily used stocks weekly. Other stocks may be subcultured every other week or monthly.

10.Diseased stocks- Diseased stocks should be subcultured every two days to dilute the disease organisms. Transfer only live beetles! Dead or moribund beetles should be discarded.

Eggs may also be collected on Gold Medal flour (or any flour which has been pre sifted through a #50 sieve), and a new stock begun from the debris-free eggs. Allow the adults to lay eggs on the fine flour for 24 hours. Each day, collect the eggs by "double sieving". This method involves using two sizes of sieves, a #25 and a #50, stacked one on top of the other with a receiving pan on the bottom (The #25 is on the top). Adults remain on the #25 sieve and can be placed temporarily in a covered, sterilized petri dish. The eggs will be retained on the #50 sieve, and can then be transferred to a clean glass petri dish. (Alternately, if the two sieves are warped and difficult to separate after sifting, egg collection can be done in two separate siftings: separate the adults from the flour using only the #25 sieve first, then sieve the flour again with the #50 sieve to collect the eggs.) All extraneous material (frass, debris) can then be removed from the collected eggs using a small brush. Put cleaned eggs in jar or bottle of fresh flour for development. (This works for ridding a stock of mites as well as disease).

B. TROUBLE SHOOTING

If a stock is not producing progeny, check the following:

1. Are there any adults still alive? If there are live adults, check to see if they are all males (some disease seems to plug up and kill the females first).
   
   
2. Is there evidence of disease. . . dead, dried, and sometimes darkened, larvae and pupae? ("Licorice stick" is a good description the of dead larvae's appearance. Dead pupae appear discolored and mummified, and are often chewed on by the adults.) 
   
   
3. Are there mites in with the adults, or clinging to the adults? To differentiate between grain mites, psocids, and parasitic mites, you can look at this web page* to see what grain mites and psocids look like.

Parasitic mites tend to hang all over the adults, sometimes to the point of giving them a frosted look, and also hide under the wings and elytra. They seem to prefer female beetles, possibly as a way of being near eggs which they may feed on.

A permanent or long term cure is a lot of work initially. A subset of adults need to be cleaned. This means putting them on ice and removing the mites with a vacuum probe or aspirator. When they are mobile and dry, put them in fine flour with topping for egg collection. Collect the eggs 1-3 days later (depending on the number of adults ovipositing). Now come the hardest part. Put the eggs in a glasspetri dish on some dark paper or other good-contrast surface, under the microscope. With an insect pin and a small vial of ethanol, remove EVERYTHING that is not a plump, healthy egg. Dip the head of the pin in the ethanol, and then blot up the trash. Swish or shake the dish and repeat. You have to be careful of mites that are feeding on the eggs, as they swell up almost egg-size, and the egg wraps around them as it is depleted. Put the now "sterile" (mite free) eggs in new flour and hope for the best. Freeze out your equipment for 24 hours.

C. TROUBLE PREVENTION

1. Keep all containers of beetles or culture flour closed or covered when not being used or worked with.
   
   
2. Bang pans and sieves vigorously against wastebasket lid before and after each use.
   
   
3. Wipe off and rap spoons and scoops against table top before each use.
   
   
4. "Squeegee" sterilize brushes before each use.
   
   
5. If beetle adults, larvae or pupae are found on the table top as a result of sieving, discard unless you saw it fall and are 100% certain of its origin! (It helps to begin with a spotless working surface and floor. This helps increase the probability of an accurate recovery of a dropped or spilled beetle. It does not insure against accidentally picking up a "fly-in" in place of the intended beetle!)
   
   
6. Don't house beetles in airtight containers, and don't push corks tightly into mouths of vials. Insects need fresh air!
   
   

D. Disease & Mites

Eggs may also be collected on Gold Medal flour (or other equally fine flour) , and a new stock begun from the debris-free eggs. Allow the adults to lay eggs on the fine flour for 24 hour periods of time. Each day, collect the eggs by double sieving. This method involves using two sizes of sieves, a #25 and a #50, stacked one on top of the other . The #25 is placed on top, with the #50 between the #25 and the receiving pan. Adults remain on the #25 sieve and can be placed temporarily in a covered, sterilized petri dish. The eggs will be retained on the #50 sieve, and can then be transferred to a clean petri dish. (Alternately, if the two sieves being used are warped and difficult to separate after sifting, egg collection can be done in two separate siftings: separate the adults from the flour using only the #25 sieve first, then sieve out the eggs using only the #50 sieve.)   All extraneous material (frass, debris)  can then be removed from the collected eggs using a small brush. Put cleaned eggs in jar or bottle of fresh flour for development. This works for ridding a stock of mitesas well as disease.

Parasitic mitescan easily retard or destroy an otherwise healthy stock. The mites hang all over the adults, sometimes to the point of giving them a frosted look. They seem to prefer females. A permanent or long term cure can be achieved, with a lot of work.

1. Initially, a subset of adults needs to be cleaned. This means putting them on ice and removing the mites with a vacuum probe or aspirator. Mites are persistent, and can also hide (safely)  under the elytra.
2. When the beetles are recovered, put them in fine flour with topping for egg collection.
3. Collect the eggs 1-3 days later (depending on the number of adults ovipositing) .
4. Now come the hardest part. Put the eggs on some dark paper or other good-contrast surface, under the microscope. With an insect pin and a small vial of ethanol, remove EVERYTHING that is not a plump, healthy egg. Dip the head of the pin in the ethanol, and then blot up the trash. Swish and repeat.
5. Be careful of mites that are feeding on the eggs, as they swell up almost egg-size, and the egg wraps around them as it is depleted. Also look for loose mites roving the surface. Roll the eggs over.
6. Put the now "sterile" eggs in new flour. Keep infested cultures and healthy cultures separate. Disinfect your equipment.

E. Developmental Rates of Tribolium castaneum

(The reproductive lifetime is 3-4 months for females and 4-6 months for males. Isolated males have been known to live for up to a year)

Note: At 22°C, development is much slower.

F. Sexing Tribolium

Separating the sexes is necessary in order to run a number of genetics tests. Both adults and pupae can be sexed. If the intended cross must be a virgin cross, it is necessary to sex the beetles as pupae to insure no previous mating has taken place. Following are some materials and methods which have worked well in our laboratory, plus suggested alternate materials you might use.

1. Pupae (Sexing beetles as pupae rather than as adults is easiest since pupae move very little compared to the adults, and do not need to be immobilized by cooling them on ice.) 

a. Materials (Microscope, light source, working surface, manipulating tools)

Microscope : A stereoscope is needed to sex the pupae. You will want to be able to magnify the pupae by at least 20 - 30X. A zoom lens stereoscope is very handy.

Light source : A good light source will reduce eyestrain if you are going to be looking at many pupae. The best is a fiber optics light system. It is both a cooler light source than conventional lights, and those on gooseneck pipes can be aimed at exactly the area you need to focus on. We use a fiber optics light with two light pipes. If you use a standard light, be careful not to overheat your pupae by having the light source too close to them.

Working surface : A small plate of a non-static generating material (approx. 3" x 4")  is very handy for separating the sexes. We use a 3" x 4" piece of styrofoam backed posterboard. This has a thickness of about 1/4" which makes it easy to pick up, is light weight, and has a smooth surface to work on. We have chosen a deep blue color, since that color provides a good contrast to the color of the pupae. Any dark color will do. Light colors should be avoided because they cause a glare from the lights.

Manipulating tools : A small natural bristle brush can be used to move the pupae on the "plate". Alternately, a commercial or a homemade vacuum probe can be used to manipulate the pupae. We use a version available through the Jensen Tool catalog, which is hooked up to the vacuum system in our building. (The same probe could also be connected to small electrical vacuum pump) . A much simpler version can be made from a plastic drinking straw, a 2-foot piece of flexible rubber tubing (approx. 1/8" internal diameter) , and a plastic pipette tip. In this case, the vacuum is supplied by the user's mouth.

Other : Plastic petri dishes or other small containers can be used to temporarily hold the pupae both before and after sexing. These same containers, or small bottles or vials which contain about 1"of flour can be used to hold the pupae until they eclose to adulthood. Any container used for this purpose should have a lid which would keep the wandering adults from escaping. (The lid also needs to have small air holes placed in the top if it is a very tight fitting lid. Petri dish lids do not need air holes.) A plastic funnelis handy for pouring pupae or adults from a sorting pan into a bottle or jar.



b. Methods



1. Tilt dish and tap some onto the sexing plate.
2. Using a small brush or vacuum probe, line up the pupae in a horizontal line about half way down the plate (have them all facing the same direction, i. e. all heads up or all heads down) .
3. As you look at each pupa under the microscope to determine the sex, brush one sex into a new line above the original line, and the other sex into a new line below the original line. When this process is done, you should have two new lines in place of the old one; one with males and one with females. Use the diagrams at right to identify males and females. (Tip: Ignore the two pointed structures at the very end of the pupa - these are the urogomphi, not the genital papillae. The female papillae, which are much larger than those of the male, are two finger-like structures just anterior to the pointed urogomphi. The male papillae are enough smaller that they look like just fingertips rather than fingers.) .
4. Double check your work. To do this, it is important to brush one of the sexes off of the plate into a petri dish or other container while you double check the other sex. (Pupae can't walk, but they can roll or squirm, and if you leave both sexes on the plate while double checking them, one could squirm into the wrong line or group, undoing your careful sexing!)  Look at each pupae again to verify its sex, placing missexed pupae in the correct group. You might want to check your pupae a third time just to be sure while you're getting the hang of it.
5. Label your containers and place each sex in a separate container with flour to allow them to eclose to adulthood. (You can use them for crosses once the adults have darkened to brown.) .

2. Adults

a. Materials (ice bucket, ice block)

Ice bucket: Any container which can hold crushed ice can be used to precool adult beetles. The small styrofoam boxes or buckets which are used for picnics or fishing are perfect for this. .

Ice block: This is used to keep the beetles immobile while you are looking at them under the microscope. We use small, flat plastic tissue culture bottles which we fill with water about 1/4"at a time until they are mostly full. (Don't fill any container completely full because it will crack or burst when frozen!)  Any low-profile container which can hold crushed ice could be used; for instance, a large petri dish full of crushed ice. The pre-cooled beetles are then placed in a smaller, low-profile container (such as a smaller petri dish lid) , and this smaller container of beetles is placed on the larger low-profile container filled with ice. You should be able to place this assemblage under your stereoscope. The beetles should remain immobile long enough for you to be able to sex them. (Tip: The ice eventually melts, allowing the beetles to "wake up", so you will want to limit the number of beetles you sex at onetime to a number compatible with the "staying power" of your cooling equipment.) .

b. Methods

1. Collect adults from stock (using methods mentioned above in Wrangling Tips)  and place in a covered petri dish or other container for temporary holding. Put the container of beetles on the crushed ice in the ice bucket to precool them before putting them on the ice block.
2. Tap a small number of adults from the petri dish into a smaller flat container on the ice block.
3. Line up the adults as with the pupae above, and separate them into two new lines according to sex. Use the diagram at right to distinguish between males and females. The males have a small patch of short bristles on the inside of the first pair of legs, about 1/3 the distance out from the bases. If the patches have picked up flour, they will appear like two domes of flour or flour paste, and will be fairly easy to see. If they have not yet picked up flour, they will appear as slightly darker, textured spots on the legs. (Changing the angle of light or changing the position of the beetle can help make the patches more visible if you are having trouble seeing them.)
4. Recheck the sexes while still on the ice block.
5. Brush each sex into a separate petri dish or other covered holding container until all the beetles are sexed.
6. (Reminder: Work with only a small number of beetles at a time. This will allow you to do the sorting and rechecking before the ice block starts to melt and the beetles wake up and try to walk off!)
7. Use the beetles for your crosses. If they are sexed as adults, they are usually used right away rather than being held in flour like the pupae mentioned above. If you do place them in containers for use later, remember that the females are probably already fertilized and will be producing offspring in their jar.