Anurida granaria (Nicolet, 1847) is a cosmopolitan species of Collembola. Specimen collected by Graham East in and around Christchurch, New Zealand, were observed to exhibit bioluminescence when disturbed. In order to enable this behavior to be observed and to make the species available to schools and universities throughout New Zealand, methods were develop for their collection, rearing and handling. Although these techniques may be utilized by experts, they are equally adaptable to students and teachers who would like to use this species in the classroom. The fact that A. granaria is a small, harmless and relatively easy to rear species, one which does not transmit disease to crops or trees and which is not a vector for human disease, this makes it an ideal bioluminescent species for classroom study. Given also that A. granaria has a wide distribution, pure stock may be safely be distributed throughout New Zealand without any harm to the ecosystem. Add to this the fact this species exhibits bioluminescence, and it becomes evident that it is an excellent species to offer teachers, students and researchers. It is therefore hoped this methodology will stimulate an interest in the species and its wide usage throughout the scientific and educational community.
Initial collection of A. granaria was made by Graham East in the Christ Church area on South Island, New Zealand. Mr. East had known of the species for many years, having chanced to observe large glowing colonies under bricks, logs and other ground cover.
When specimens were collected and sent for analysis it was found that these natural colonies consisted of a mixed group of species. Therefore it became necessary to individually and carefully pick out those individuals which were exhibiting bioluminescence. This was done with great patience and skill using mouth operated aspirator to secure glowing specimens from those of a group.
Specimens so collected were preserved in alcohol and sent for analysis. It was thus established that A. granaria was the species which was bioluminescent. This determination was made by Wanda M. Weiner Ph.D,Wanda M. Weiner Ph.D., Head of the Laboratory of Invertebrates at the Institute of Systematics and Evolution of Animals in Krakow, Poland.
Live specimens were placed in containers for rearing and maintaining pure stock.
Because A. granaria is cosmopolitan, specimens collected in the field were a mix of different species. Once this was established, A. granaria was carefully isolated, selecting only for bioluminescent individuals. Then from these individuals pure stock was established.
Although this may sound like an easy task, it certainly was not. A. granaria is a very tiny animal measuring approximately 1.4 mm in length. The only way to distinguish A. granaria from other species is careful microscopic examination of preserved specimens by an expert. That is, in daylight the individuals from this mixed group of Collembola are difficult to distinguish one from the others, especially if you are a novice. One individual may, to the untrained eye, look very much like another. Therefore it is necessary to test each individual for bioluminescene. This selection must be done in darkness, using an aspirator to collect only those Collembola producing light. Another technique which proved very successful was to sort Collembola in the light, placing each individual in a small 12 mm x 75 mm test tube. Then examine test tubes under total darkness at night. When a test tube containing A. granaria is sharply tapped one will see a brief glow. Hence one may readily separate the glowing species, A. granaria, from species which do not glow. One may also maintain specimens inside the test tubes to double check selection. Using this method it is possible to weed out Collembola which do not exhibit bioluminescence, leaving only bioluminescent A. granaria. After learning to distinguish A. granaria in this manner, the careful observer may better collect them from a mix in the field and find that when tested for bioluminescence a greater ratio of A. granaria to species which do not exhibit bioluminescence is obtained (this is according to field collection results Graham East has told to the author in private correspondence).
Over time significant numbers of bioluminescent individuals were collected and isolated from which pure stock was obtained. This became very apparent for when one has a container of A. granaria it only takes a gentle blowing upon them to cause bioluminescence. Also one then finds that all individuals are similar in every aspect of their appearance and expert analysis of a representative sample shows that only one species is present.
Because obtaining a pure stock in this manner is a difficult, time consuming process which requires the assistance of an expert Collembola taxonomist, it is recommended that those wishing to obtain A. granaria simply order a pure stock. This avoids duplication of effort, saves time and money and is really the most sensible way to obtain A. granaria now that a pure stock has been established.
As reported by Michael L. Draney of the Dept. Natural and Applied Sciences, University of Wisconsin-Green Bay, Collembola can be reared on a moist substrate and fed yeast. Moist soil or peat moss works adequately. For better results, Draney says one may use a plaster of Paris/powdered charcoal substrate kept moist with aged tap water. Small plastic or glass jars provide with tight fitting lids are the recommended rearing vessel. Collembola may be fed powdered baker's yeast and watered added at least once per month.
In keeping with these recommendations it was found that A. granaria do indeed thrive upon moistened peat moss or a plaster of Paris/powdered charcoal substrate when fed yeast. A primary consideration may be maintaining a pure stock free of species of Collembola which are normally found with A. granaria.
A good procedure is to place stock rearing jars inside plastic rearing boxes set inside a cabinet. These may be stacked and lighted with a fluorescent lamp. This type of arrangement enables easy and safe handling of stock. When A. granaria are kept on a period of 12 hours of light and 12 hours of dark, they then may be observed to exhibit bioluminescence only during the dark period.
Also pure stock must be kept free of mites. In order to establish and maintain pure stock care must be used to begin only with specimens which are of a single species, i.e., A. granaria. In this case that was achieved by selecting only bioluminescent individuals to use in establishing stock. To insure that stock was only of a single species, a single large, bioluminescent adult was selected, washed and introduced into a rearing vessel with substrate. This was repeated to provide greater odds of isolating a single adult fertile female. Once these females produced young, they were reared. The stock was checked to insure that no mites were present and that only A. granaria was present. Over time this process was repeated to insure that the resulting stock was a singular species and free of mites.
When rearing A. granaria or any species of Collembola for that matter, it is best to use great care to avoid introduction of mites from the very start of one's rearing program. Because mites may occur in samples of soil where cosmopolitan Collembola occur, one should NOT use a Berlese funnel to establish stock, as obviously mites would be collected. The better method is careful collection of singular individuals as above stated. Yet even after establishing a culture on a sterile substrate one should check for mites. This may be done using floatation. When a sample of substrate is flooded with water and left 24-48 hours, mites float to the top and may easily be seen, collected and observed under a microscope. When one uses this method it is easy to establish whether or not a stock has been established which is is free of mites. It is recommended that in effort to maintain pure cultures this method be used to regularly check for mites and insure that stock does not become contaminated.
Given the small size and fragility of A. granaria, it is recommended that when establishing new stock cultures or when handling specimens, they not be brutalized by rough handling. This is especially true if you want to risk injury to specimens intended for experimental purposes. One way to transfer specimens from one vial to another is to use the response of A. granaria to light, gravity fermenting food. A. granaria will move away from light and down toward a fermenting food source. As seen in the above figure this is an easy way to transfer specimens from one jar or vial into another.
This method of transfer may also be used to collect individuals or groups of individuals into test tubes, Petri dishes or other tiny chambers or vials for experiment without touching or damaging specimens. Other methods employed to pick up and transfer specimens about include the usage of an aspirator, red sable artist's brushes, wire loops and an medicine eye dropper. Given A. granaria float upon water, this may be employed to aid in their safe manipulation.
The observation and photography of the bioluminescence of A. granaria presents a challenge owing to the low intensity of light emission and the fact that this must be observed after stimulating A. granaria by vibration or sound produced in absolute darkness or under very low light levels. One method proposed is to place A. granaria in small observation cells. These may be made in any number of ways. Given that other Collembola have been long reared upon potato, making observation cells out of potato may prove to be a promising methodology in this regard. Another idea is to make observation cells using clear plastic soda straws which may be secured to the stage of a microscope, concentrating a number of A. granaria into a small area using potato plugs as plungers.
A. granaria are an ideal species for teachers to use in the classroom to demonstrate bioluminescence in insects. All that is required is to observe A. granaria in a perfectly darkened room. By gently blowing upon A. granaria they will briefly glow. When this is seen for the first time by young and old alike, it results in an impressive feeling of awe and amazement. What better way to encourage an interest in science and learning that using A. granaria to demonstrate bioluminescence and stimulate the learning process!
A. granaria are also ideal for a variety of experiments. For example, one may maintain stock under different periods of light and dark to determine how this effects bioluminescence. It should be found that when kept under a 12/12 daylight/dark period, A. granaria will only exhibit bioluminescence during the dark phase. This circadian rhythm may prove to be an interesting topic for further investigation.
A. granaria is also ideal for studying the effect of diet or other variables upon bioluminescence. Simply add different chemicals to the substrate upon which A. granaria feed and see what happens. For example does the pH of a substrate, variation in diet or addition of other chemicals effect bioluminescence?
Students just learning how to maintain pure cultures may wish to experiment to discover which type of glassware or other vials are most useful in rearing stock. Considerations here are how to handle A. granaria so as to maintain pure stock free of such pest as mites or fruit flies.
Certainly the observation and photography of the bioluminescence of A. granaria presents a challenge. Students may try to devise their own methodology in this respect with the objective to observe and photography A. granaria under a microscope when it glows. Another challenging area for experiment is to devise new substrates upon which to rear A. granaria. In this regard the author has developed MARS2020 which may prove to be greatly superior to the standard plaster of Paris-charcoal mix generally used by those who rear Collembola. The MARS2020 substrate may be made commercially available in the future.
Students and researchers alike may wish to investigate the mechanism of bioluminescence. Can simple experiments be devised to prove the nature of bioluminescence in A. granaria? This certainly represent a challenge, especially if presented to students giving them no clue as to the nature of bioluminescence. Let students dwell upon the mystery and see what they suggest in the way of experiments to seek an answer to this perplexing question.
A. granaria represents a great addition to the teacher's arsenal of species to use in the classroom and in the laboratory. For this reason alone schools and universities far and wide would be wise to obtain and maintain stock of A. granaria. If you are interested in adding A. granaria to your repository of teaching tools, please place your order today. Thank you.
