Conotoxins: a Case for their Cause of the Rapid Evolutionary Radiation of
Conus
Andy Malishenko
Colorado State University
Fort Collins, Colorado 80523
4/22/94
INTRODUCTION
Cone snails are one of the most diverse and dominant predators in
the coral reef. Their recent appearance and rapid speciation is enough
to catch any body's eye interested in stories of adaptive radiation.
In this paper I will propose and support that the rapid evolution of
cone snails is due to their unique venom.
CONE SNAIL BIOLOGY
Cone snails of the genus Conus inhabit tropical waters at varying
shallow depths. They get their name from the distinctive conical shape
of their shell. The designs on the shells are beautifully intricate and
some shells are worth a small forture (See Figure 1). The greatest
diversity of cone snails can be found in subtidal choral reefs of
Thailand and Indonesia but their geographic range extends throughout the
Indio-Pacific region (Kohn 1975, Olivera 1990). They are nocturnal
predators of a remarkably broad phylogenetic range of prey including
polychaete worms, hemichordata, cephalopods, other gastropods, bivalves,
fish, and Crustacea (Nybakken 1990 Miller 1989). They have a very
specialized feeding mechanism involving the use of a hollow tooth at the
end of the proboscis used to stab and envenomate prey (See Figure 2 and
3) (Kantor 1990). The toxins in the venom act very rapidly. The fish
hunting snail, Conus prupurascens can paralyze its prey in about two
seconds (See Figure 1) (Olivera 1991). This quick action is especially
important if you are a snail trying to catch a fish! The mollusc hunter
Conus textile's venom induces peristaltic convulsions in its prey.
Again useful as many molluscs tend to retreat into their shell upon
attack. The convulsions apparently prevent this from occurring
(Woodward 1990). Let's take a close look at the evolution of the cone
snails.
PHYLOGENY AND EVOLUTION
Cone Snails are molluscs of Class Gastropoda, Order Neogastropoda,
and Superfamily Toxoglossa. Toxoglossa contains the families
Terebridae, Turridae, and Conidae. The specialized mode of feeding
described above is exclusive to the Toxoglossa. Most, but not all, of
its members feed in this way (Kantor 1990). The only living members of
the Family Conidae are Conus species. Distinctions between Turridae and
Conidae are very vague and some taxonomists include both in Conidae.
Taxonomic classification of the Toxoglossa varies depending on who is
writing the paper but the above is not iconoclastic (Kohn 1990).
The first Cone Snails appeared after the Cretaceous-Tertiary
boundary approximately 55 million years before the end of the
Pleistocene epoch (Kohn 1990). Current counts of species in the genus
Conus are around 500, making it perhaps the largest single molluscan
genus (Olivera 1991). Figure 4 shows a kite diagram of Conus species.
You can see an expansion of the genus through the upper Miocene where
species number drops as we enter the Pliocene. This reduction in
species number of most gastropods and other invertebrates is
characteristic of this epoch (Raup 1976). Then very rapid speciation
occurs in the Pleistocene leading to the recent count of 500 (Kohn
1990). Geographic radiations occurred as well, expanding their
geographic range from Coastal European origins to the Indio-Pacific
region (Kohn 1990).
The striking feature of Conus evolution is the large number of
species contained in the single genus that only appeared since the
extinction of the dinosaurs. Attempts to explain this rapid
evolutionary radiation fall generally along two lines. Did Conus find a
new adaptive zone to invade or did some "key innovation" (usually
morphological) occur that helped them succeed (Kohn 1990)? As with most
adaptive radiation stories, the answer is unclear. The answer to
weather Conus entered a new adaptive zone is difficult to say. The main
evidence for this would come from the fossil record which has so far not
proved to be very elucitory. I feel that the key innovation hypothesis
warrants a closer look as these are no ordinary venomous snails. Their
venom is very unusual.
CONUS VENOM
The venoms of the cone snails contain short peptides called
Conotoxins. Each species' venom contains a mixture of these toxic
peptides along with other molecules. Figure 5 shows an HPLC analysis of
a peptide fraction from Conus Magnus venom. One can see the multiple
peaks and the biological activity induced in mice by each of theses
fractions (Olivera 1991). In their active form, these peptides consist
of only 10 to 30 amino acids. They have a highly conserved cysteine
"backbone" which provides for multiple cross-linked disulfide bonds as
the peptide folds. Many animals have toxic peptides with disulfide
cross-linking but none are as short as the Conotoxins (Olivera 1991) .
Conotoxins are originally translated into propeptides of intermediate
length. The cysteine residues, however, are concentrated near the
COOH-terminus. Once the protein has folded, the N-terminus is cleaved
and only the short, cysteine rich end of the peptide remains. Figure 6
illustrates the difference between conotoxins and other cysteine-rich
peptide toxins (Olivera 1990). The cleaved N-terminus of conotoxins is
believed to be involved in assisting the folding of the peptide (Olivera
1990 Woodward 1990).
Over 75 peptides from 10 venoms have been biochemically
characterized with others being sequenced monthly. Currently there are
known to be three basic cysteine structural frameworks into which most
of the conotoxins can be categorized. (There are some Conus toxins
without disulfide bonding but they are not as well characterized)
(Olivera 1991). These categories are distinguished by the position of
the cysteine residues and the number of loops formed in the folded
peptide as seen in Figure 7.
The inter-cysteine residues of different conotoxins belonging to
the same category are quite variable. In fact, some believe they are
hypervariable, denoting that there may be a special genetic mechanism
used by the cone snails to create increased variability of these inter-
cysteine residues (Woodward 1990). Further study needs to be done to
elucidate the mechanism for this observed hypervariability.
Within the major structural categories there are proposed
subgroups based on the general makeup of the variable amino acids.
Figure 8 show how the "four-loop" structural category can be broken down
into more specific groups based on the charge of these variable inter-
cysteine residues. This is the most well studied category of
conotoxins.
One more note about the sequence of these peptides involves the
cleaved N-terminus region. As I mentioned before, this is thought to be
involved in controlling the folding of the peptide. One would expect,
therefore, that the sequence of this region would be highly conserved
within a group of conotoxins. As Figure 9 shows, three peptides of the
King-Kong group exhibit this sequence conservation.
CONOTOXIN TARGETS
These toxins exhibit their poisonous effect by blocking specific
ion channels of nerve cells. Figure 10 shows the specific channels that
the three cysteine rich categories of conotoxins affect.
This channel specificity is quite remarkable. Many drugs used for
medical purposes bind their desired target but also bind related targets
which often produce unwanted side affects. In contrast to most drugs
conotoxins can discriminate among closely related receptor subtypes
(Olivera 1990). For example drugs such as dihydropyridine that is used
to block calcium channels will bind such channels in smooth, skeletal,
and cardiac muscle and well as neuronal tissue. Omega-conotoxins bind
only to the neuronal calcium channels. One can easily see how this is a
benefit to the cone snail. If its main goal is paralysis, it doesn't
want to waste its toxin by having it bind to channels irrelevant to the
paralysis of its prey. The other categories of conotoxins show this
degree of specificity as well. Mu-conotoxins discriminate between
muscle and neuronal sodium channels. Alpha-conotoxins block
acetylcholine receptors only at the neuro-muscular junction and not
inter-axonal (Olivera 1990).
WHAT A GREAT DESIGN
To sum up all of this toxin business, we have a genus who's
species produce toxins of unsurpassed specificity for behavioraly
relevant targets in the nervous system of their prey. These species
have a constrained mechanism of producing toxins which enables them to
create many variations on a theme. Within a structural category, the
rigid cysteine backbone and conserved folding apparatus combined with a
hypervariability of inter-cysteine residues results in the ability of
cone snails to produce a plethora of related peptides. This way, new
toxins can be produced that may have new specificities without risking
complete loss of biological activity. This strategy would be
particularly useful if the targets of the toxins were related as well
(Woodward 1990). Well that is exactly the case with ion channels. The
evolutionary relatedness of ion channels is shown in Figure 11 from
Hille 1992.
The specificity of conotoxins is even more specific than this
diagram reveals but it still illustrates the relatedness of the ion
channels. In summation, cone snails have developed a scheme for
designing a variety of toxins that are very likely to be effective to
their ends of prey capture. They have not only produce effective, quick
acting toxins, they also have a mechanism ideal for designing new
peptides targeted to the nervous system of their prey.
WHERE'S THE BEEF
The literature shows that cone snails have undergone rapid
speciation and have expanded their geographic range in a relatively
short (geologically speaking) period of time. I proposed that this was
due to a key innovation, namely the conotoxin venoms. I have shown why
I think the conotoxins are very ingenious and bound to (in my opinion)
bring about success but I haven't given any proof to support my claim
that the venoms are responsible for the success of Conus. That is what
this next section is about.
TEST OF VENOMS AS KEY INNOVATION
In order for an innovation to be responsible for rapid
evolutionary radiation, it must meet several criteria. (Adapted from
Herera 1989 in Kohn 1990)
1. Taxa with the feature diversify early in their evolutionary history.
2. The novel feature is significant to the taxon and absent from its
sister or ancestral taxon.
3. Taxa with the feature become structurally and taxonomically more
diverse than sister taxa lacking it.
The diversification of the Conus genus has occurred over the past
55 million years. To date, every Conus species' venom that has been
characterized has contained peptide venoms. This tends to support the
hypothesis that the peptide venoms have been around since the species
origins. If this were not the case, we might expect to see other genera
in the family without peptide venoms. Therefore we can conclude that
the peptide venoms are possible candidates for a key innovation
responsible for their rapid evolutionary radiation.
Since the venom is important for prey capture by cone snails, it
is obviously significant. As for the rest of criterion number two, we
must first decide what are the sister and ancestral taxa of Conus. The
most closely related genus to Conus would be contained in the family
Turridae (Miller 1989). It is not known weather this family is sister,
ancestral, or neither (Kohn1990). Reguardless, most believe there is
some relationship between the two families so a comparrison is
appropriate. Unfortunately, the nature of the venoms of the genera in
Turridae is not known (Kantor 1989). Further study of the Turrids
would help to answer this question.
In satisfying the third criteria, the genus Conus has more species
that any Turrid genus (or almost any molluscan genera for that
matter)(Kay 1990). Also the food types of Conus include polychaete
worms, hemichordata, cephalopods, other gastropods, bivalves, fish, and
crustacea whereas the entire family of Turridae is limited to polychaete
worms, sipunculida, nemertea, and other molluscs (Miller 1989). This
shows higher diversity among the cone snails. Also, the cone snails
have morphologically different hollow teeth which correspond to
differing prey types (See Figure 12) (Nybakken 1990). This has not been
shown in any Turrid genera.
So that is the beef. If you were hoping for a quarter pounder I
bet you were disappointed. The truth of the matter is that there is not
much known about the Turridae to provide a good comparison with Conus.
Hopefully in the not so distant future we will have answers to some of
the important questions (such as the nature of Turrid venoms and their
feeding physiology) that will shed some light on this ambiguous yet
still interesting story.
DEVILS ADVOCATE
Even if you grant that the above criteria are met for conotoxins
being a key inovation in Conus, this does not mean that they are
responsible for their rapid evolutionary radiation. It may be some
other morphological feature or something completely different. Again,
more study is needed to support the main thrust of this paper.
CONCLUSION
Conotoxins are some of the most efficiently designed poisons known
to man. They are proving to be a valuable tool in medical research.
Due to this fact, much funding is available for research on conotoxins
and the literature is expanding as you are reading this. Because
scientist like science and not only the money they get to study, I
believe we will learn much more about these toxins than just their
pharmacological value. There is a very interesting adaptive radiation
story hidden in the sequences of the DNA of these small (but definitely
not innocuous) cone snails which is just waiting to be unraveled by some
curious scientist.
BIBLIOGRAPHY
Cruz L.J., Ramilo C.A., Corpuz G.P., Olivera B.M. Conus Peptides:
Phylogenetic Range of Biological Activity. Biological Bulliten. N183
p159-64 August 1992.
Herrera C.M. Seed dispersal by animals: a role in angiosperm
diversification? American Naturalist. V133 p309-22 1989.
Hille B. Ion Channels of Exitable Membranes, Second Edition. Chp 20
1992.
Kantor Y.I. Anatomical Basis for the Origin and Evolution of the
Toxoglossan Mode of Feeding. Malacologia V31 N1 p3-18 1990.
Kantor Y.I., Sysoev A.V. The Morphology of Toxoglossan Gastropods
Lacking a Radula, with a Description of New Species and Genus of
Turridae. Journal of Molluscan Studies. V55 p537-49 1989.
Kay E.A. Turrid Faunas of Pacific Islands. Malacologia. V32 N1 p79-87
1990.
Kohn A.J. Tempo and Mode of Evolution in Conidae. Malacologia. V32 N1
p55-67 1990.
Kohn A.J., Nybakken J.W. Ecology of Conus on Eastern Indian Ocean
Fringing Reefs: Diversity of Species and Resource Utilization. Marine
Biology. Marine Biology V29 p211-34 1975.
Miller J.A. The Toxoglossan Proboscis: Structure and Function. Journal
of Molluscan Studies. V55 p167-81 1989.
Nybakken J. Ontogenetic Change in the Conus Radula, Its Form,
Distribution Among the Radula Types, and Significance in Systematics and
Ecology. Malacologia. V32 N1 p35-54 1990.
Olivera B.M., River J., Clark C., Ramilo C.A., Corpuz G.P., Abogadie
F.C., Mena E., Woodward S.R., Hillyard D.R., Cruz L.J. Diversity of
Conus Neuropeptides. Science V249 p257-63 1990.
Olivera B.M., River J., Scott J.K., Hillyard D.R., Cruz L.J.
Conotoxins. The Journal of Biological Chemistry. V266 N33 p22067-70
1991.
Raup D.M. Speceis Diversity in the Phanerozoic: a tabulation.
Paleobiology. V2 p289-97 1976b.
Photograph of shells from Science. V230 p1341 1985.
Woodward S.R., Cruz L.J.., Olivera B.M., Hillyard D.R. Constant and
hypervariable regions in conotoxin propeptides. The EMBO Journal V9 N4
p1015-20 1990.