| Deutsche Sprache | Snap-tentacles & microscope | Artikel und Berichte |
 HOME
|
 HOME |
|
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. Enzyme production 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
Results
The results of the new battery of experiments are as follows: a. All nine Drosera species showed unequivocal positive
results.
Conclusions
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 Symbols used - - -
= 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) Bibliography 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 |