Download COMMENTARY Chemical or nematocyst

COMMENTARY
Chemical or nematocyst-based defence in the nudibranch Cratena
peregrina? – a reply to B.K. Penney
Arnaldo Marin
Departamento de Ecologı´a e Hidrologı´a, Facultad de Biologı´a, Universidad de Murcia, 30100 Murcia, Spain
Correspondence: A. Marin; e-mail: [email protected]
(Karuso, 1987; Rogers & Paul, 1991). Thus, metabolite
accumulations within opisthobranchs may represent a mechanism to store toxic chemicals with impunity for subsequent elimination, and not necessarily to defend the organism (Pennings
& Paul, 1993).
Penney suggests that because E. carneum from North
American possesses chemical extracts that deter feeding by
pinfish, extracts from Eudendrium employed with models of C.
peregrina could be unpalatable because they contain chemicals
and not active nematocysts. Stachowicz & Lindquist (2000)
indicated that crude extracts from Eudendrium deterred feeding,
‘demonstrating’ that defensive chemistry plays a significant role
in the unpalatability of these species. To test their hypothesis
that unpalatable secondary metabolites represent alternative
antipredator defences for hydroids, they compared the palatability of intact polyps with those in which the nematocysts
had been chemically stimulated to discharge. Hydroid polyps
defended solely by nematocysts should become palatable when
their nematocysts are discharged before offering them to predators, whereas the palatability of chemically defended hydroids
should not change after treatment. However, neither the lipophilic (DCM þ butanol soluble compounds) nor the watersoluble fractions of Eudendrium deterred feeding. These authors
suggested that there is an additive or synergistic effect among
compounds in the two fractions, or that the deterrent compounds decomposed during the partitioning of the crude
extract. In my opinion these results do not demonstrated that
defensive chemistry plays a significant role in the unpalatability because toxins contained in nematocysts could also act as a
chemical deterrence. If there truly are active chemicals in
Eudendrium, the lipophilic and the water-soluble fractions must
be clearly deterrent to fish, but this is not the case. Secondary
metabolites contained in the discharged nematocysts could be
responsible for fish rejection.
The biosynthesis of defensive metabolites is costly for organisms. It is not surprising that species showing de novo biosynthesis of defensive metabolites use these metabolites for other
purposes. This is the case in the nudibranch Tethys fimbria,
which synthesizes prostaglandin derivates (Marı́n, Di Marzo &
Cimino, 1991). The structural variety of the lactones and the
data on their distribution in the body of T. fimbria suggest a
range of different biological functions: to contract smooth
muscle fibres, defence allomones of the ceratal secretion, and in
basic physiological functions (e.g. ion regulation, renal function
and reproductive biology). These arguments do not mean that
other aeolidoidean nudibranchs should also contain defensive
metabolites. The aeolid nudibranch Phyllodesmium guamensis
feeds on the soft corals Sinularia maxima and S. polydactyla.
Phyllodesmium guamensis sequesters a diet-derived diterpene
11b-acetoxypukalide selectively within various body parts.
Levels were highest in the cerata, with moderate and low to
non-existent levels in the mantle and viscera, respectively.
Laboratory tests showed that 11b-acetoxypukalide deters
feeding by the puffer fish Canthigaster solandri at a given concentration (0.5% dry mass), while feeding field experiments with
Journal of Molluscan Studies (2009) 75: 201–202
# The Author 2009. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved
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Aguado & Marin (2007) analysed the interaction between
Cratena peregrina (Gmelin, 1791) and predatory fish in laboratory and field assays, using both live aeolids and artificial
models. Penney’s comment (2009) on the article reopens the
old question of the role of nematocyst-based defence in nudibranchs. Penney suggests that the defensive mechanism of
C. peregrina is chemical because the nematocysts used in
assays could be unarmed. Penney argues that (1) Aguado &
Marin (2007) obtained nematocysts by macerating Eudendrium
hydroids with a mortar and pestle, and this cannot be considered equivalent to kleptocnidae isolated from C. peregrina;
(2) the method by which the authors attempted to incorporate
nematocysts into the test food is unclear; and (3) regardless of
how nematocysts were added to the artificial food models, it is
possible that no functional nematocysts would remain by the
time fish encountered them.
The main purpose of our article was to demonstrate that fish
learn to avoid the warning coloration pattern of C. peregrina,
due to associating bad taste or unpleasant experience with the
colour pattern. Nematocysts are contained in the defensive
exudates and in the external part of the body of C. peregrina,
which can only be regarded as circumstantial evidence that
these defensive cells or the molecules that they contain represent a deterrent for predators. Of course, the article does not
provide experimental evidences that the source of deterrence is
the nematocysts, but there is no proof, either, that chemical
defences play an active role. If we support the general idea
that a positive result for deterrence alone cannot be taken as
evidence for a nematocyst-based defence, the same argument
must apply to chemical-based defence. We must classify
nematocyst-based defences as a special form of chemical
defences because nematocysts contain harmful molecules. From
this point of view, C. peregrina transfers chemicals from food to
the cnidosacs at the tip of the ceras.
There is no evidence that C. peregrina contains secondary
metabolites other than the nematocyst toxins. Chemical analysis supports the general hypothesis that the nematocysts are the
main weapons. Cimino et al. (1980) studied three species of the
hydroid Eudendrium (E. rameum, E. racemosum, E. ramosum) in the
Bay of Naples (Italy), which are prey of C. peregrina. These
authors found the same pathway of polyhydroxylated steroids
(Cholest-4-en-4, 16b, 18, 22R-tetrol-3-one 16,18-diacetate) in
the hydroids and in the nudibranchs. Unfortunately these
steroids do not play a defensive role in hydroids or nudibranchs. Some aeolids contain pigments and secondary metabolites from their prey, which probably have no chemical
defence role either (e.g. Goodwin & Fox, 1955; McBeth, 1972:
the compounds are ubiquitous and conserved amongst numerous non-deterrent species; Cimino et al., 1980: the compounds
are located within internalized structures and thus not quickly
encountered by predators). Sequestered compounds can occur
at concentrations lower than threshold feeding deterrent levels
COMMENTARY
nudibranch tissues exhibited species-specific reactions to the
nudibranch tissues and their isolated metabolite, which ranged
from non-deterrence to emesis (Slattery et al., 1998).
Even if active deterrent metabolites are present in C. peregrina,
it is very unlikely that chemical defence is the sole defensive
mechanism, because retaining functional nematocysts must be
energetically expensive. Several studies in opisthobranchs have
indicated that chemical and physical defences commonly
co-occur and can function either additively, or synergistically,
to reduce susceptibility to predators. In the nudibranch Doris
verrucosa, several defensive strategies occur at the same time.
This nudibranch is perfectly mimetized in its habitat and its
mantle is protected by spicules and by the presence of two toxic
molecules, verrucosin-a and -b (Cimino et al., 1988). Many
algae and invertebrates display redundant defence mechanisms
against predation (Hay et al., 1994; Schupp & Paul, 1994).
Nudibranchs utilize both physical (spicules, tunic toughness)
and chemical (secondary metabolites, acidity) defences and
suffer relatively little predation by generalist predators.
The method by which we incorporated nematocysts into the
test food was clearly described: ‘the artificial models were
bathed in hydroid sauce’ (Aguado & Marin, 2007). We used
the word impregnation incorrectly because it suggests that the
hydroid sauce entered and spread completely through the artificial food. The Eudendrium sauce was applied as a coat to the
outside of models, because if nematocysts were mixed with the
artificial food before solidification the high temperature could
have degraded them. This method was chosen because nematocysts are not released into the cnidosac of the aeolid, but into
large epithelial cells called cnidophages. The release of nematocysts from aeolids is caused by a contraction of the muscle
complex surrounding the cnidosac, and entire cnidophages are
released (Greenwood & Mariscal, 1984). These cnidophages
are forced towards the ceras tip where they are pushed into a
cellular matrix and through the epithelium, before being
released from the ceras. The application of isolated nematocysts
to artificial food might be considered inappropriate, because it
does not reflect the defensive mechanism of aeolid nudibranchs. The coating of artificial models with hydroid sauce is
more realistic, because it simulates the expulsion of the entire
cnidophage in the aeolids, rather than a bioassay with isolated
nematocysts. It is also suggested that any nematocysts that
might have adhered to the models could be washed off when
the models came into contact with the water of the aquarium
or field site and that it is possible that no functional nematocysts would remain by the time fish encountered the models.
Penney’s criticism is in error, because our personal observation
indicates that a high percentage of nematocysts remains
undischarged in the models. However, just a small percentage
of active nematocysts might be sufficient to deter fish in the
bioassays with artificial models. In addition, Martin (2003) in
a study of the management of nematocysts in C. peregrina found
that large proportions of these nematocysts were undischarged
in the alimentary tracts of the snails. This author even
describes that the faeces from snails contained large numbers
of undischarged nematocysts that appeared structurally intact.
Nematocysts were isolated from the sea anemone Calliactis
tricolor maintained under different feeding schedules or in
different degrees of salinity, in an attempt to determine how
these culture conditions influence the discharge of isolated
nematocysts. The nematocysts isolated from individuals from
two separate populations of C. tricolor responded significantly
differently to 5 mM EGTA and to de-ionized water, and these
responses also depended upon the isolation solution used. This
study provides evidence that the environment or feeding state
of an anemone affects the discharge of isolated nematocysts.
Inherent differences in ionic and osmotic characteristics among
nematocysts could explain some of the ambiguities when
comparing past studies of isolated nematocyst discharge
(Greenwood et al., 2002).
Penney also suggests that nematocysts obtained by macerating
Eudendrium cannot be considered equivalent to kleptocnidae isolated from C. peregrina. However, Martin (2003) in an exhaustive
study of C. peregrina found, when feeding on polyps of the hydrozoan Eudendrium, that phagocytosis of nematocysts and food particles by digestive cells is rather non-selective. In the course of
over two years of this study there was no evidence of selective
exclusion of specific nematocyst types by cnidophagous cells.
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doi:10.1093/mollus/eyp007
Advance Access Publication: 20 March 2009
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