Carnivory in Byblis
liniflora revisited (I) : A simple method for enzyme
testing on carnivorous plants
Introduction and theory
At the beginning of this year I had a conversation with Dr. Jan Schlauer from the University of Würzburg (Germany) concerning carnivorous plants (CPs). In the course of which he mentioned a report by Heslop-Harrison and Knox (1971), in which the authors describe an easy method to detect enzyme activities in carnivorous plants using photographic film. This seemed so interesting to me that I decided immediately to try this out. The following day I spoke to an acquaintance in Switzerland (Luigi Francini), whose hobby is photography and who has a small photographic laboratory. I asked if he would be willing to develop some film for me. Luigi Francini (who also has some CPs at home) was enthusiastic, and after a quick lesson about different materials used for photography the first tests started the following weekend.
The theory is quite simple. Digestion enzymes dissolve the gelatine layer of exposed film. The gel contains the finely distributed photosensitive chemicals such as halogenous silver crystals, pigments, etc., and is applied as an extremely thin suspension onto the plastic substrate which is mostly made from acetylcellulose or polycarbonate. Strips of processed but not fixed Kodalith ortho (ASA 12) black & white film are used for the test.
The enzyme production of the plants is triggered by yeast-solution. Twelve hours later some dexterity is required to fix the film strips for 24 hours onto the trapping leaves. After removal twenty-four hours later, the strips show a typical spotting if enzymes are present. Laboratory examination of enzymes (e.g. with protein gels) is very expensive whereas this procedure with customary films is a very simple alternative, is very low-priced (really only 1 or 2 cents per test !!) and is thus really interesting for use in private greenhouses and even for field tests if accordingly adapted.
Suitable photographic material and its application
As mentioned above, Kodalith ortho (black and white) film is used in the literature, mainly for lithography, microphotography and other industrial purposes because it is very robust and provides a wide range of contrast. It is an industrial film which usually must be specially ordered by photo shops. Since it was not easily available, I decide to test some different types of customary material with varying gelatine-layers. Luigi Francini recommended three different types of ILFORDä black & white films. The more photosensitive this film is, the thicker it is and the more coarse grained its halogenous silver crystals are. A series of tests were used to show which material was most suitable. ILFORDä FP4 (ASA 125), ILFORDä HP5 (ASA 400) and ILFORDä XP2 (ASA 400) were tested on Drosera capensis, Drosera cuneifolia and Byblis liniflora. Instead of the method described in literature, I used only exposed (and not additionally processed and unfixed) material because it was my idea to find a test which was practicable and easy as possible. To stabilise the results on the strips after the exposure to the plants for further photographs, they were processed but not fixed by Luigi Francini and afterward showed finer and durable details. Significant clearer results could be seen on the two coarse grained ASA 400 negatives (which had the thicker gelatine layer). Some looked like ‘X-ray’ images so the test plant could often be identified, whereas the FP4 ASA 125 was much lower in quality (where in one case a Drosera cuneifolia fed with a mosquito larvae digested a 2 mm hole into the gel). Even after processing, the negatives of the sundews show only a few, poor spottings and Byblis liniflora showed nothing. This result is surprising to me, because the Kodalith ortho film used in literature has only a sensitivity of ASA 12.
The negatives of the HP5 and XP2 showed the enzyme activities exactly the other way around, meaning black and white in reverse. Held against the light, the XP2 showed the clearest outlines of glue drops and (in excellent cases) even of the tentacles, so that it displayed nearly a complete image of the trapping leave. The colour of the whole leaf area is light to pale grey, perhaps caused by soaking of the gelatine or by leaching soluble parts of the layer by humidity during the exposure on the plant. It was not a result of enzyme activity because the same effect also took place with pure water. More interesting were the black centres with clear perforations. Here the gel was dissolved by enzyme. The light shining through those holes in the layer and the black zones around provide a good contrast. For this reason the ILFORD XP2ä is chosen as the most suitable film.
To expose the film I simply pulled it out of the roll and cut it into 1.0 to 2.5 cm broad stripes, according to the size of the plant to be examined. To mark the film type and assigned plant durable (and in a way that would not be obliterated by the processing-bath) I cut different notches into the strips. Dates were conscientiously recorded. As mentioned above, correct fastening of the film strips on the trapping leaves of CPs needs a little imagination to achieve good " illustrations" !
Choice of suitable CPs
To check the method, I chose adult plants of Drosera capensis and Drosera cuneifolia as reference, because the enzyme production of sundews (Drosera) has been thoroughly examined and proven. In addition I use a one-year old rainbow plant (Byblis liniflora) of which the production of enzymes was still unclear. Bruce (1905) did some investigations on Byblis gigantea, growing near Perth in Western Australia that showed positive results, but did not examine Byblis liniflora. This question of carnivory for Byblis liniflora was of special interest to me since I had visited some north Australian habitats with my wife Irmgard, and we demonstrated a mutualism involving Capsid bug species (Miridae) and Byblis liniflora on our video ‘Beautiful and Hungry Part 2 CP’ (1995). That symbiosis showed much similarity with the two species of the South African Roridula and their Pameridea-bugs, which are also true bugs belonging to the Miridae. We could observe this symbioses for six years in our living-room in Weil am Rhein, and got some nice video shots which can be seen on our video ‘BEAUTIFUL AND HUNGRY - CP’ (1994). However, a number of experts do not include Roridula with CPs because it shows no enzyme production. If the tests proved that Byblis does not produce enzymes and also shows the same symbiosis with closely related Capsid bugs, a new discussion by the experts about the assignment of the Rainbow Plant as a CP would be necessary.
This line of thought is particularly interesting because of the current discussion about the plumbagin branch of CP. Plumbagin is a compound present in the " old" CP families Droseraceae, Drosophyllaceae, Nepenthaceae and Dioncophyllaceae in which all species are able to produce proteolytic enzymes (Schlauer 1997). Observing the ‘plumbagin-free’ branches, only the two " modern" CP families Cephalotaceae and Lentibulariaceae show this uniform behaviour.
All other families have at least members which do not produce enzymes. Even the Sarraceniaceae, which are the stars of nearly every CP-book, contains such plants, e.g. Darlingtonia and several species of Heliamphora. Interestingly, without any own enzymes those plants make numerous bacteria and arthropods do the digesting. This was confirmed recently by Ellis and Midgeley (1996), who did large-scale tests with isotope-labelled (15N) flies on Roridula associated with Pameridea-bug populations. They observed that the isotopes sucked out of the prepared insects by the resident Capsid reached the plant leaves via the feces dropped on the leaves, and were being absorbed probably through stomata. So it has been proven that Roridula is able to benefit directly from the wide selection of prey sticking to its leaves and not only via soil fertilisation by the natural decay of it’s prey. The results of further enzyme tests can contribute to the clarification of the question of carnivory and whether in future some of those plants assigned today as true CP (e.g. Byblis, Darlingtonia, Heliamphora), may not be classified carnivorous anymore, or if the definition for a true CP must be reformed to allow enzyme-free, and passive pre-digestion by symbiotes.
In the literature, a yeast solution is used to trigger the enzyme production by the plants. Some tests with pure yeast, as well as solutions diluted by water, all show a clear reaction from Drosera, which meant the tentacles and sometimes the whole leaf started to surround the treated spot. If the solution is too concentrated coarse lumps appeared on the traps. This resulted in low quality illustrations of the tentacles and glue drop contours on the film. Pure yeast and dried mosquito larvae (which show the strongest reaction !) yielded similar results. The best results achieved a 10 % solution of yeast in water. Depending on the trap size, 1 - 3 drops were applied using a small pipette. This procedure resulted in the most detailed outlines and was digested by all plants without problems.
Preparing the series of tests
The tests described above yielded a second result concerning Byblis that I didn’t expect to be so unequivocal. On ASA 400 material all Drosera appeared as clear positive, but no Byblis showed enzyme production. To explore this result (which is surprising for what is usually considered a true CP), and to further test the whole laboratory procedere, I decided to follow the first tests with the following experiments:
a) Nine further different Drosera species as additional positive references. See table 1
The results of the new battery of experiments are as follows:
a. All nine Drosera species showed unequivocal positive results.
Using species of Drosera as a reference is recommended. The results of the test on Byblis liniflora are a real surprise. Further experiments on other CP species are in preparation, but to describe them would go beyond the scope of this article. The described tests are not only very inexpensive (1 film roll allows approximately fifty tests !), they are very easy to perform, show good reproducibility and are absolutely harmless for the examined plants. In fact, the more yeast or mosquito larvae are given to the plants, the more they benefit. These attributes make the test highly interesting for private CP collectors, and so (I hope) promise a flood of new test results if published for a wider, CP- interested, public. Easy field experiments are possible if the tested plants are sheltered against the weather during the exposition time.
A highlight of this study is the following: If a sensitive (e.g. ASA 400) conventional black and white film is used in the above described way, a reproducible and detailed low cost test without any chemical bath is possible!
Last but not least I would like to express my gratitude to Luigi Francini for his friendly support and professional treatment of all photographic works. And I wish success for everybody who tries the test, because I’m sure we can await further surprises on this subject.
Table 1: Test Results
Results of the enzyme experiments performed 17 January 1997 - 26 January 1997
- - - = Enzyme test negative+ = Enzyme test positive (Gelatine layer shows dark centres without perforation)
++ = Enzyme test positive (Gelatine partially perforated)
+++ = Enzyme test positive (Gelatine layer strongly perforated, possibly big holes)
Bruce, A.N. (1905) On the actuvity of the glands of Byblis gigantea,
Notes Royal Botanical Garden, Edinburgh, 16: 9-14.
Carow, T. (1996) Todesfallen oder Lebensspender ? Die neue Sicht der fleischfressenden Pflanzen (documentary film). In Radke R, (ed.),
ZDF (German TV) ‘Naturzeit’, Thomas Carow Nüdlingen (Germany).
Ellis, A.G., and Midgley, J.J. (1996) A new plant animal mutualism involving a plant with sticky leaves and a resident hemipteran insect, Oecologia, 106: 478 - 481
Hartmeyer, S., and Hartmeyer, I. (1994/95) Beautiful & Hungry - Part 1 & 2 Carnivorous Plants, private video production, contact the author of this article.
Heslop-Harrison, Y., and Knox, R.B. (1971) A cytochemical study of the leaf-gland enzymes of insectivorous plants of the genus Pinguicula, Planta 96: 183 - 211
Juniper, B.E., Robins, R.J., Joel, D.M. (1989) The Carnivorous Plants, Academic Press Limited, London.
Schlauer, J. (1997) " New" data relating to the evolution and phylogeny of some carnivorous plant families, Carnivorous Plant Newsletter, 26: 34 - 38