Chem. Senses 28: 141-153,
2003
© Oxford University Press 2003
RESEARCH PAPERS |
Inducible Defences in Daphnia Depend on Latent Alarm Signals from Conspecific Prey Activated in Predators
1 Department of Natural Sciences, Faculty of Mathematics and Sciences, Agder University College, Kristiansand, Norway 2 The Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway 3 Present address: Department of Biology, University of Mississippi, University, MS 38677, USA
Correspondence to be sent to: Dr Ole B. Stabell, Department of Natural Sciences, Faculty of Mathematics and Sciences, Agder University College, Serviceboks 422, N-4604 Kristiansand, Norway. e-mail: ole.b.stabell{at}hia.no
Abstract
Some water fleas (Daphnia spp.) undergo phenotypic changes when exposed to chemical signals from predators. The chemical signals have been assumed to be of predator origin (i.e. kairomones), since juices of crushed Daphnia have been found ineffective. We speculated that latent alarm signals could be present in Daphnia, to be activated in predators following ingestion. Accordingly, fish predators were fed earthworms for 10 weeks to remove Daphnia remains from their gastro-intestinal tracts. Following another 6 days of earthworm feeding, water conditioned by fish induced no morphological changes in D. galeata. When fish were alternatively fed Daphnia for 6 days, changes were induced with fish-conditioned water. Extracts made from intestines of earthworm-fed fish, homogenized with earthworms, gave no morphological changes, but intestines of the same origin homogenized with Daphnia did. Similar results were found when earthworms and Daphnia were homogenized with fish liver. Freshly frozen extracts of homogenized Daphnia gave no detectable changes at first instar stage in test animals, whereas extracts of Daphnia that had been kept at room temperature did induce such changes. Our results suggest that Daphnia respond to latent conspecific alarm signals (i.e. `dormant' pheromones) that are activated by intestinal or bacterial enzymes in predators or in the water.
Key words: fish odour, growth, kairomone, morphology, phenotypic changes, pheromone
Introduction
By the end of the nineteenth century, it had become clear that the seasonal
variation in appearance of waterfleas (Daphnia spp.) observed in
lakes and ponds was not due to the succession of many species, but resulted
from seasonal intraspecific changes in morphology (i.e. cyclomorphosis). A
`floating-theory' was proposed to explain the variation in Daphnia
morphology during changing seasons
(Wesenberg-Lund, 1900
).
According to this theory, Daphnia develops enlarged spines and
`helmets' as floating devices to counteract increased sinking rates associated
with warmer summer waters. Many investigators opposed the theory and several
alternative hypotheses have later been proposed to explain the phenomenon of
cyclomorphosis (Jacobs,
1987).
Dodson (Dodson, 1974)
observed that the morphology of Daphnia varied as a result of
predation and suggested that the changes that took place during cyclomorphosis
were adaptive. Rotifers (genus Brachionus) had at that time been
shown to develop protective spines as a response to chemical cues released by
Asplanchna, a rotifer predator
(Gilbert, 1966). In
Daphnia, however, the importance of chemical cues associated with
predation was not recognized until some years later, when two papers appeared
simultaneously (Grant and Bayly,
1981; Krueger and Dodson,
1981). In the first paper, a predator-induced `helmet' was
reported in individuals of the D. carinata complex
(Grant and Bayly, 1981). The
changes in these animals were induced by a chemical stimulus released by the
invertebrate notonectid predator Anisops calcaratus. The authors
suggested that crest development was a predation-avoidance mechanism, with the
crested morphs being better at escaping predator attack. In the second paper,
Krueger and Dodson (Krueger and Dodson,
1981) reported `neckteeth' in Daphnia pulex embryos
following exposure to chemical cues released by the predatory midge larva
Chaoborus americanus. Their results also suggested that the induced
form experienced reduced mortality, implying that the neckteeth of
Daphnia were probably a defence against predators. The superiority of
crested morphs compared to typical morphs of D. pulex in escaping the
grasp of predatory Chaoborus larvae has later been confirmed in
behavioural studies (Havel and Dodson,
1984).
Following the two initial reports, morphological changes induced by
chemical stimuli have been reported for a number of Daphnia spp. The
shifts have been found with chemical cues from both invertebrate predators and
fish (Hebert and Grewe, 1985;
Havel and Dodson, 1987;
Dodson, 1989;
Vuorinen et al.,
1989; Walls and Ketola,
1989; Hanazato,
1990,
1991; Tollrian,
1990,
1993,
1994;
Lüning, 1992;
Tollrian and Dodson, 1999).
The morphological changes caused by chemical cues from predators are commonly
termed `inducible defences' (Harvell,
1990; Tollrian and Harvell,
1999).
Tail spine length is one morphological trait that varies during changing
seasons in D. galeata
(Primicerio, 2003). For that
species, the timing of increase in tail spine length corresponds with the
seasonal dietary switch in stickleback, the main fish planktivore in many
lakes at high latitudes. Spaak and Boersma
(Spaak and Boersma, 1997) have
shown that chemical stimuli associated with fish induce the elongation of the
tail spine in D. galeata, as previously shown for other congeneric
species (Tollrian and Dodson,
1999). The defensive roles of tail spines of Daphnia with
regard to small fish predators has been demonstrated
(Kolar and Wahl, 1998).
In addition to the morphological changes mentioned above, Daphnia
also display behavioural and life-history adaptive responses to predators
(Larsson and Dodson, 1993;
Boersma et al., 1998;
Tollrian and Harvell, 1999).
Following early findings (Dodson,
1988) of chemically induced predator-avoidance in
Daphnia, predator-induced behaviour has later been found to be
triggered by chemical cues from a number of invertebrates and fish predators
(Ringelberg, 1991;
Dawidowicz and Loose, 1992;
De Meester, 1993;
Watt and Young, 1994;
Kleiven et al., 1996;
Lauridsen and Lodge, 1996;
De Meester and Cousyn, 1997;
Stirling and Roff, 2000).
Responses in life-history traits, such as size and age at first reproduction
and the production of males and resting eggs (i.e. ephippia), have also been
found triggered with predator-associated stimuli
(Dodson and Havel, 1988;
Ketola and Vuorinen, 1989;
Machácek, 1991,
1993,
1995; Lüning,
1992,
1994,
1995;
Stibor, 1992;
Weider and Pijanowska, 1993;
Stibor and Lüning, 1994;
Tollrian, 1995;
Pijanowska and Stolpe, 1996;
Burks et al., 2000).
It has previously been implied that the various response types (i.e.
behavioural, morphological and life-history changes) may result from the same
set of chemical cues (Ketola and Vuorinen,
1989; Lüning,
1992; Tollrian,
1995; Ringelberg and Van Gool,
1998). It may therefore be helpful to consider functional
properties released by chemical cues for all three types of responses
combined.
Chemical signals that are detected by a prey organism may have several
sources. The signals may be interspecific messengers, originating in the
predator or another prey species and termed `kairomones'
(Brown et al., 1970).
Alternatively, the signals may have their origin in injured conspecific prey.
In that case, they function as intraspecific alarm substances—i.e.
`Schreckstoff' (Pfeiffer,
1963)—and should be classified as `pheromones'
(Karlson and Lüscher,
1959). With regards to inducible defences in Daphnia, the
active chemical cues have generally been considered as kairomones since juices
of crushed Daphnia have been found ineffective
(Walls and Ketola, 1989;
Parejko and Dodson, 1990).
Additional support for the view that the active compounds do not originate in
conspecific prey comes from the fact that the signals appear to possess
predator-specific properties (Dodson,
1989; Stibor and Lüning,
1994).
Alarm signals have been demonstrated in a number of aquatic animal species
(Pfeifer, 1963; Howe and Sheik,
1975; Atema and Stenzler,
1977; Parker and Schulman,
1986; Smith, 1992;
Wilson and Lefcort, 1993).
With respect to predator—prey interactions in aquatic environments, it
was long assumed that prey animals were detecting chemical cues of predator
origin. However, several studies of prey behaviour, carried out with careful
control of the predators' diet, have revealed that predators may be `labelled'
with chemical alarm signals from previously ingested prey. Predator labelling
has been shown in prey species as diverse as sea anemones, fish, amphibians,
insect nymphs, marine snails and sea urchins
(Howe and Harris, 1978;
Mathis and Smith, 1993;
Wilson and Lefcort, 1993;
Chivers et al., 1996;
Jacobsen and Stabell, 1999;
Hagen et al., 2002).
Predator labelling has also been demonstrated with regard to morphological
defences induced by alarm signals in fish
(Stabell and Lwin, 1997).
The concept of predator labelling postulates that a predator is being
chemically disclosed by its choice of prey, but remains undetectable by
distance chemoreception as long as its diet does not include recognizable prey
(Howe and Harris, 1978;
Mathis and Smith, 1993;
Stabell and Lwin, 1997). Alarm
signals from ingested prey are probably released from the digestive system of
predators together with urine and faecal material
(Wilson and Lefcort, 1993).
Therefore, to distinguish alarm signal responses from true kairomone responses
requires experimental evidence from research with proper control for predator
diet.
We speculated that chemical cues from predators (i.e. kairomones) must be
regarded as `unreliable' with regard to predation risk for the prey. This is
because the intensity of fish predation on Daphnia in a lake is not
constant, but varies seasonally due to phenological changes in fish diet. The
smell of fish can only inform Daphnia of fish presence, not of fish
diet. Another objection to the reliability of predator `kairomones' as cues of
predation risk stems from evolutionary considerations. Predators that release
easily detectable chemical cues might be expected to end up as losers in the
long run. It can therefore be argued that the development of an advanced
defence system in a prey, based on predator cues, does not represent a
plausible evolutionary scenario (Parker,
1984). Intriguing support for our speculations comes from a survey
of the literature. In almost every paper dealing with defences in
Daphnia that are supposedly induced by predator kairomones, the
predators were fed the prey species of study.
Based on the knowledge listed above, we expanded the idea of how predator labelling may take place and proposed the presence of latent alarm substances in Daphnia that require activation by digestive enzymes or bacteria within the gastrointestinal tract of the predator. Such chemicals would provide reliable signals for the assessment of predation risk by Daphnia. Here we present evidence for the presence of such latent alarm signals in Daphnia.
Material and methods
Collection and rearing of animals
The water fleas (Daphnia galeata) used in the experiments were
collected from Lake Lombola, located in the inner part of Troms County, North
Norway (69° 07' N). Another cladoceran prey (D. pulex) used
in some treatments was collected from a pond (<100 m2) on the
main island of Tromsø. Clonal lines of D. galeata were reared
at room temperature in the laboratory, kept in a synthetic zooplankton medium
made from glass-distilled water with 0.5 g unrefined salt and 0.1 g
CaCO3 per litre (Hobæk
and Larsson, 1990). The salt was heated to 450°C for 12 h,
dissolved and filtered through glass fibre filters (Whatman GF/F), before the
final dilution. A mixed stock of D. pulex was reared in 20 l glass
jars, using filtered stream water from the college campus. A freshwater green
alga (Scenedesmus acutus) was used as food for the
Daphnia.
Three-spined sticklebacks (Gasterosteus aculeatus), a native
predator fish, were trapped in a stream pond on the college campus and Malawi
cichlids (Nimbochromis venustus), an alien predator fish, were
obtained from an aquarium shop in Tromsø. The fish were fed either one
of the two Daphnia species, or earthworms (Lumbricus spp.),
depending on the experimental protocol. The rearing of sticklebacks took place
in a cold-room at 
6°C, whereas the cichlids were reared in the
laboratory at ambient room temperature. In addition, crucian carp
(Carassius carassius), raised in the aquarium facilities on
commercial fish feed and rolled oats, were used as donors of fish liver.
Preparation of stimuli
To produce predator-conditioned water, sticklebacks and cichlids were
initially fed earthworms for 10 weeks. This was done to secure removal of
possible remains from previously ingested Daphnia in the
gastrointestinal tracts of the fish. Thereafter, three groups of sticklebacks
and three groups of cichlids were each placed in separate 5 l aquaria and fed
alternatively (a) D. galeata, (b) D. pulex, or (c)
earthworms for 6 days. To produce fish-conditioned water, two fish from each
species and treatment series were allowed to swim in 3 l of zooplankton medium
for 24 h. During this period the fish were not fed and the water was
continuously aerated by airstones. Following the removal of fish, the
fish-conditioned water was filtered (Whatman No. 1) and subsequently frozen in
350 ml plastic ice-cube bags at -18°C for storage
(Stabell and Lwin, 1997). To
avoid contamination, different nets were used for each treatment to transfer
fish between the rearing aquarium and the glass container used for
conditioning of water. Short names for the various types of
predator-conditioned water used in the study are listed in
Table 1 (1a-c, 2a-c).
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Tissue extracts to be used as chemical stimuli were made in three different
ways. In the first case, Daphnia and earthworms were each homogenized
with intestines of predator fish that had been kept on an earthworm diet for a
minimum of 10 weeks (Table 1,
3a,b). Extracts were prepared by homogenizing five adult
individuals of D. galeata, or small pieces of earthworms (0.3 g)
with pieces of intestine from sticklebacks (0.2 g) in 5 ml of zooplankton
medium. Following a 2.5 h respite at room temperature to allow for bacterial
and enzymatic activity to take place, the homogenates were centrifuged. The
supernatants were diluted in a 1:100 ratio by volume with zooplankton medium
and frozen in plastic ice-cube bags. Secondly, a sample extract was made from
five adult specimen of D. pulex that were homogenized with 0.1 g
of crucian carp liver in 5 ml zooplankton medium. Extract of earthworms,
homogenized with crucian carp liver and zooplankton medium, was used as a
control sample (Table 1, 4a,b).
Also in these cases, the homogenates were left at room temperature for 2.5 h,
followed by centrifugation, dilution and freezing of the extracts until usage.
Thirdly and finally, samples were made from Daphnia homogenized with
zooplankton medium only (Table 1,
5a,b). Altogether, 10 adult specimen of D. galeata were
used and half the homogenate was immediately centrifuged, diluted and frozen.
The other half was left at room temperature for 2.5 h before centrifugation,
dilution and freezing of the stimulus sample.
Experimental protocol
All experiments were conducted with a single clonal line of D. galeata and took place at room temperature (19-20°C) under conditions of constant light in glassware that had been thoroughly washed, water rinsed and autoclaved. At the onset of each series, 100 ml of zooplankton medium was added to each glass, with five replicate glasses for each treatment. The glasses were labelled according to treatments and placed at random on the bench. Two egg-bearing individuals of D. galeata were put in each glass (with the aim of obtaining a sufficient number of offspring for each parallel) and 1 ml of zooplankton medium was replaced by 1 ml of the stimulus solution prepared for each treatment. The relevant stimulus was thereafter added once a day during the experimental period using the above replacement procedure.
Each glass was followed for 24 h to register offspring release. In general, this took place in a minimum of three glasses during the surveillance period. The 24 h limit for awaiting offspring production was set to produce homogeneous time series. Following offspring release, the mothers were removed and the developmental stages of the progeny followed. At regular time intervals after birth (days 1, 3, 5 and 7) one individual was taken at random from each replicate glass and measured. On day 1, the offspring were sampled within 12 h after birth (i.e. instar 1); by day 7, some individuals were carrying eggs (i.e. instar 5). Growth takes place stepwise in Daphnia, during shedding of the exoskeleton to establish the next instar stage.
Altogether, five series of experiments were conducted. In series 1, D. galeata were exposed to water conditioned by sticklebacks that had been fed (a) D. galeata, (b) D. pulex, or (c) earthworms. In addition, five replicates with added zooplankton medium (d), were run as blank treatments. The short names used for the exposure types of this series were `galeata—stickleback', `pulex—stickleback', `earthworm—stickleback' and `blank', respectively (Table 1, 1a-d). In series 2, exposure took place with water conditioned by three groups of Malawi cichlids that had been given the same three feed types (a—c) as in the series with sticklebacks. Also in this series, a blank treatment (d) of zooplankton medium was used. The short names for the exposure types of this series were `galeata—cichlid', `pulex—cichlid', `earthworm—cichlid' and `blank', respectively (Table 1, 2a-d). In series 3, D. galeata were exposed to extracts of intestine from earthworm-fed sticklebacks that had been homogenized with either (a) D. galeata, or (b) earthworms. The short names used in this series were `galeata—intestine' and `earthworm—intestine' (Table 1, 3a, b). In series 4, exposure took place with extracts of crucian carp liver homogenized with (a) D. pulex, or (b) earthworms. The short names used in this series were `pulex—liver' and `earthworm—liver' (Table 1, 4a,b). Finally, in series 5, D. galeata were exposed to extracts of either (a) homogenized D. galeata frozen fresh, or (b) homogenized D. galeata left for 2.5 h at room temperature before freezing. The short names used for the exposure types of this series were `galeata-fresh' and `galeata-aged' (Table 1, 5a, b).
Morphometric measurements and data analysis
Morphological measurements were carried out using a microscope equipped
with an ocular micrometer. The measured parameters were helmet length (HL),
body length (BL) and tail spine length (SL)
(Hebert and Grewe, 1985;
Hanazato, 1990;
Pijanowska, 1990). HL was
measured as the distance between the anterior margin of the compound eye and
the tip of the helmet (Figure
1). When individuals lacked a helmet, the distance measured was
between the eye and the anterior margin of the head. BL was measured as the
distance between the posterior margin of the insertion point of the second
antennae and the posterior margin of the carapace. SL was measured from the
posterior margin of the carapace to the tip of the spine. BL was measured at
40x magnification, whereas HL and SL were measured at 100x.
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To adjust for variation in size among individuals, data are presented as
`tail spine index' (SI), or `helmet index' (HI), given by the formulae SI =
SL/BL and HI = HL/BL (Hanazato,
1990; Pijanowska,
1990; Tollrian,
1990). To test for statistical differences between treatments
(a = 0.05), the data were analysed using Student's t-test.
Statistical calculations were carried out using the computer program Statview
(SAS Institute, Cary, NC).
Results
Growth patterns
To visualize the general patterns of growth in D. galeata in the presence and absence of chemical cues from predators, the absolute lengths of body, tail spine and `helmet' from hatching (instar 1) until the first observed stage of egg production (instar 5) are presented in Figure 2. Water conditioned by Malawi cichlids previously fed D. galeata was used as the predator cue in this example, whereas zooplankton medium was used as control cue. Individuals of D. galeata exposed to water conditioned by predators demonstrates a pattern of length increment for body, tail spines and `helmets' different from that obtained following exposure to control treatments. During the first 3 days, tail spines and `helmets' were found longer in the presence of predator cues compared to when the predator cues were absent. For tail spines, the differences between treatment types were found at a statistically significant level (P < 0.05) on both day 1 and day 3. From day 5 on, body lengths were found to increase in the presence of predator cues compared to control treatment, whereas tail spine and `helmets' tended to decrease. For body length, a statistically significant difference (P < 0.05) was found on day 5 with these treatments.
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Exposure to predator-conditioned water
When D. galeata were exposed to the `earthworm—stickleback' treatment (Table 1), both the tail spine and the helmet indices showed an almost horizontal pattern (slight decline) during the 7 days of exposure (Figure 3A, stippled lines). This means that the relative sizes of crests are only slightly decreasing with increasing instar stages. Almost identical patterns were found for D. galeata exposed to the `blank' treatment (Figure 3B, stippled lines). No significant difference was found between the above treatments for either tail spine or helmet indices throughout the duration of the time series (P > 0.05). This result shows that predator-odour alone is not sufficient for induction of morphological changes in Daphnia.
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When D. galeata were exposed to the `galeata—stickleback'
treatment (Figure 3A, solid
lines) or the `pulex—stickleback' treatment
(Figure 3B, solid lines),
however, another pattern of progress revealed for both indices. For both
treatments, the tail spine index started at a higher value on day 1 than found
with exposure to `earthworm—stickleback' or `blank' treatments and it
declined throughout the experimental period to end at a lower value than
obtained with those treatments on day 7. This result is in accordance with
what is found in nature in the presence of fish predators, where increase in
spine length develops for small Daphnia, followed by decrease in
spine length when the animals are approaching mature stages, i.e. instar 5 or
higher (Primicerio, 2003).
Also, the helmet index was found to be larger than controls on day 1 for the
`galeata—stickleback' and the `pulex—stickleback' treatments. The
helmet index, however, increased slightly towards day 3 for both treatments,
and subsequently decreased towards lower values than controls on day 7. For
both treatments, statistically significant differences from
`earthworm—stickleback' and `blank' treatments were found for the tail
spine index on days 5 and 7, whereas statistically significant differences
were found for the helmet index on days 1 and 3 for the
`pulex—stickleback' treatment and on day 1 for the
`galeata—stickleback' treatment. These results strongly suggest that
native predators must have ingested specimen of Daphnia beforehand to
induce morphological changes in D. galeata.
The general patterns obtained for the tail spine and helmet indices with stickleback-conditioned water were also found with water conditioned by Malawi cichlids (Figure 3C,D). No statistically significant differences were found in pattern of development between the `earthworm—cichlid' and `blank' treatments (Figure 3C,D, stippled lines). The major difference revealed with cichlid-conditioned water compared to stickleback-conditioned water, were the higher indices found for both tail spines and helmets on day 7 with water conditioned by fish fed Daphnia (Figure 3C,D, solid lines). However, direct statistical comparison is not possible between cichlid-conditioned waters and stickleback-conditioned waters, since these treatment groups result from different time series. For both the `galeata—cichlid' and the `pulex—cichlid' treatments, statistically significant differences from `earthworm—cichlid' and `blank' treatments were found for the tail spine index on day 1 and 3. This was also the case for the tail spine index on day 5 with the `galeata—cichlid' treatment. For the helmet indices, however, only the `galeata—cichlid' treatment resulted in statistically significant differences from control treatment (i.e. `earthworm—cichlid' treatment), as given on days 1, 3 and 7. The above results demonstrate that also an alien predator will induce morphological changes in D. galeata if specimens of Daphnia have been ingested beforehand.
Exposure to extracts of homogenates
When D. galeata were exposed to the `galeata—intestine' treatment (Figure 4A, solid line) or `earthworm—intestine' treatment (Figure 4A, stippled line), the observed patterns of response were almost similar to those obtained with predator-conditioned waters (Figure 3). In this case, however, more pronounced differences were obtained between treatments, revealing significant differences on days 1, 3, 5 and 7 for the tail spine indices and on days 1, 3 and 7 for the helmet indices. The larger differences displayed may in part be due to the fact that the `earthworm-intestinal' curve was slightly increasing during the surveillance period compared to `earthworm—stickleback' and `earthworm—cichlid' treatments. However, data representing these mentioned treatment curves were obtained from different time series, and different procedures for producing stimuli (conditioned water versus tissue extraction) may have influenced the result. Accordingly, the only conclusion to be drawn from these data is that extracts of predator intestinal tissue mixed with tissue of alien prey do not possess alarm signal properties. The tissue from predators must be mixed with tissue from Daphnia to release signals that trigger morphological responses.
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Exposure of D. galeata to `pulex—liver' treatment (Figure 3B, solid line) or `earthworm—liver' treatment (Figure 3B, stippled line), showed the same patterns as found with predator-conditioned waters and intestinal extracts. Significant differences between the two series were found on days 1 and 3 for the tail spine index, but no differences were apparent for the helmet indices throughout the duration of the experiment. In the series with extracts of homogenized liver, there were not enough Daphnia offspring available to obtain data on day 7 for the `earthworm—liver' treatment. However, the results from the first 5 days of the series with liver homogenates support the previous conclusion that the chemical substances in question do not originate in the GI tract of predators. Combined, the results obtained with homogenates of intestine and liver tissue strongly suggest that the active compounds should be searched for in the prey itself, i.e. in the tissue of Daphnia.
When live specimens of D. galeata were exposed to extracts of homogenized conspecifics, statistically significant differences were found on day 1 (i.e. at 1st instar) between the `galeata-aged' and the `galeata-fresh' treatments. The differences on day 1 were evident for both the tail spine and the helmet indices (Figure 3C). For both indices, the data obtained with bench-stored extract followed a pattern similar to that found for other Daphnia-containing homogenates. However, the data obtained with the `galeata-fresh' treatment displayed a shift in the pattern, from a start similar to that generally found with `blank' treatments on day 1, to a pattern similar to that found with bench-stored extracts containing Daphnia from day 3 on. This result reveals that the fresh extract was initially inactive, but displayed active signal properties after > 12 h in the water.
Discussion
Daphnia galeata, the test species of the study, displayed a crest
increment in the early instar stages of their life when exposed to predator
signals. For later instars, body growth was given priority, presumably to
prepare for egg production. The morphometric patterns for tail spine and body
length displayed in our data are in accordance with results from field studies
and their ecological implications are treated in detail elsewhere
(Primicerio, 2003). In this
study, fish predators were used initially to mimic the production of the
active chemical signals that hitherto have been termed `kairomones' in the
literature. This was done by feeding fishes with earthworms for 10 weeks,
resulting in loss of active chemical cues responsible for induction of crest
development in Daphnia. When fed Daphnia, however, predators
again released chemical cues with active signal properties. In this way also,
an alien fish species was labelled by prey specific signals, the result being
the release of active chemical signals by a presumably unknown predator.
The active chemical signals were subsequently made by homogenizing Daphnia with various types of tissues from predators. Both intestines and liver, taken from fish that previously had been fed earthworms, were found to release active chemical signals when homogenized with Daphnia. When homogenized with earthworm tissue, similar fish tissues displayed a lack of active cues. Even Daphnia homogenized alone were found to contain active chemical signals, but in all cases the homogenates needed a 2.5 h incubation time to release the active signals.
Freshly made homogenates of Daphnia were initially found inactive when tested for crest induction in D. galeata. The fresh extract, however, displayed a shift in signal properties after some hours in the water. This shift in pattern of crest induction suggests that inactive compounds were present in the extracts of freshly homogenized Daphnia and were chemically altered in the water after >12 h, resulting in active substances that triggered prey responses. Bacteria present in the rearing water may have caused such a shift in chemical properties of the extract.
In the following, the origin of the novel chemical signals will be considered and the necessity of pre-treating predators to detect latent alarm signals will be addressed. The possible ways in which signal activation may take place will be evaluated and the implication for explaining species specificity of signals released by various predators will be presented.
The origin of signals
One initial concern in this study was to ensure that any possible
Daphnia remains were removed from the predators. Accordingly, a
thoroughly pre-treatment of the predators was carried out to ensure that they
were not labelled by chemical cues presumably present in Daphnia. The
pre-treatment of predators was carried out by feeding fish for a prolonged
period of time with taxonomically distant prey (i.e. earthworms). A lack of
predator pre-treatment may help explain the conflicting results obtained by
other investigators. For instance, Dodson (Dodson,
1988,
1989), found in behaviour
experiments that Daphnia responded only to those predators that had
been a source of Daphnia mortality in nature, whereas the effect of
predator signals on morphology was less conclusive. In addition, Loose et
al. (Loose et al.,
1993) found in studies of vertical migration that it did not
matter whether the predator fish was hungry, fed Daphnia or fed
artificial food; the tested water was positive in all cases. However, our
results suggest that the previous feeding of predators could represent a
serious source of error and that predators can be made undetectable in an
alarm signal context by controlling their feeding history.
For cyprinid fishes, it has been found that the alarm signals from one
single club cell, diluted in 180 l of water, can be detected by conspecifics
(Smith, 1992). Due to such low
sensory thresholds in prey animals for detection of alarm signals, a complete
removal of Daphnia residues from the intestine of predators should be
a matter of concern in experimental design. The time needed to remove chemical
signals of prey origin should be expected to be longer than that needed for
the bulk passage of food through the intestine of a predator. Hypothetically,
a predator could even be marked for life following consumption of a single
prey individual. This could be the case since chemical signals may possess
hydrophobic properties (Stabell
1987). Prey odour could then be stored in the adipose tissue of a
predator, to leak slowly into the environment over time. Intermediate
hydrophobic properties of the chemical signals affecting Daphnia have
already been demonstrated (Parejko and
Dodson, 1990; Tollrian and Von
Elert, 1994; Von Elert and
Loose, 1996; Von Elert and
Pohnert, 2000).
Stirling (Stirling, 1995)
considered the possibility of predator labelling, but ended up rejecting the
hypothesis. However, the conclusion was based on an experimental design that
involved repeated switching of food sources, with a risk of undesired
labelling in the treatments. Since, otherwise, predator labelling has not been
a concern in studies with Daphnia, the time needed to remove
Daphnia residues from a predator has not been studied. However, by
feeding fish predators for at least 10 weeks with earthworms in the current
study, a complete removal was obtained with regard to chemical alarm cues. In
this way it was shown that fish predators must eat Daphnia in order
to release the active chemical signals, implying involvement of alarm signals
of prey origin and predator labelling as the functional mechanism. We suggest
that similar results will be found also with invertebrate predators if these
are subjected to a proper pre-treatment procedure.
In many aquatic animal species, vertebrates as well as invertebrates,
behavioural and morphological responses to predators cues have been reported,
whereas conspecific alarm signals are seemingly absent. In light of the
results of the present study and the subsequent discovery of latent alarm
signals in sea urchins and marine snails
(Hagen et al., 2002;
Jacobsen and Stabell, 2003),
we suspect that latent alarm signals may be a common phenomenon among aquatic
animals.
Possible mechanisms of signal activation
The basic idea behind this study was that digestive enzymes in the gut of
predators act on engulfed Daphnia tissue to activate latent chemical
signals. Parejko and Dodson (Parejko and
Dodson, 1990) suggested that the chemical cues necessary to induce
`neckteeth' in D. pulex originated in the intestinal tract of the
predator. Our data demonstrate that intestinal tissue of predators contain
neither active signals nor any chemical precursors. However, when intestinal
tissue from earthworm-fed predators was homogenized with Daphnia,
then active chemical signals were found to be present. In fact, intestinal
tissue was not required, since active signals were also obtained when
Daphnia was homogenized with enzyme rich tissue such as fish liver,
or even when homogenized Daphnia tissues were left alone. In all
cases, homogenates required incubation time to ensure that activation of the
latent alarm signals had taken place.
It is interesting to note that a total absence of predator influence on the
activation of latent alarm signals would make it impossible for the predator
to mask its presence, making the chemical signals evolutionarily reliable. How
then, does the activation take place? Homogenized material was left at room
temperature for 2.5 h to allow enzymes to act, but this procedure was also
open to bacterial influence. Evidently, bacteria were abundant in all types of
material used, especially from within the gut of predators. Freshly frozen
extract of homogenized Daphnia was activated after being introduced
into the water, releasing a response in Daphnia after >12 h.
Bacteria in the water seem the most likely candidates for such signal
activation. However, under natural conditions, signal activation should be
expected to take place within the gut of a predator. Ringelberg and Van Gool
(Ringelberg and Van Gool,
1998) found that the behavioural responses of Daphnia to
water conditioned by perch was significantly decreased if the fish was treated
with the antibiotic ampicillin and suggested that bacteria were the true
source of kairomones. Our findings give support to the idea of bacterial
involvement, but the data reveal that bacteria can only be mediators in the
activation process.
Earlier experiments, reporting various effects of exposure to extracts of
crushed Daphnia, can be interpreted in light of our new findings.
Walls and Ketola (Walls and Ketola,
1989) tested juices of crushed D. pulex in morphological
experiments with negative results. Fresh extracts were added to new jars daily
and the animals were transferred, while number of neckteeth was counted. By
this procedure, sufficient bacterial activation of latent cues from
Daphnia in the water may have been obstructed. It is interesting to
note, however, that the crushed Daphnia treatment used by Walls and
Ketola (Walls and Ketola,
1989) gave moderate responses in some of their series. The data
provided by Pijanowska and Kowalczewsky (Pijanowska and Kowalczewsky, 1997b)
also seem to support the idea that bacteria in the water may activate latent
alarm signals. In that study, too, the growth medium was changed daily, but a
significant effect of crushed Daphnia was observed from instar VI on.
Further, Parejko and Dodson (Parejko and
Dodson, 1990) tested for neckteeth development in D.
pulex and reported a lack of effect from extracts of conspecifics.
However, their fresh extracts were subjected to an extensive filtering
procedure with a final cut-off at a mol. wt of 500 Da (Herbert and Grewe,
1985). The signal precursors, which must be assumed to be larger in molecular
size than the active signals, may simply have been removed by the filtering
procedure. Slusarczyk (Slusarczyk,
1999) also prepared fresh media daily when testing for production
of ephippial eggs in flow-through chambers, but no responses were found with
crushed Daphnia alone. However, when crushed Daphnia was
added in combination with water conditioned by fish (fed chironomids), similar
responses to those obtained with water conditioned by Daphnia-fed
fish were detected. In this case, bacteria from fish in the water may have
accelerated the activation of latent alarm signals from Daphnia.
Stirling (Stirling, 1995)
prepared the chemical cue by grinding a portion of D. galeata,
followed by dilution in spring water. The water was then mixed into the
observation chambers containing D. galeata. The vertical distribution
of individuals in the water column was then followed for 1 h, but no
behavioural effects were observed. In that case, the time lapse between
production and use of homogenates may have been insufficient for bacterial
activation to take place in the water. On the other hand, Pijanowska
(Pijanowska, 1997) reported a
significant difference in vertical distribution after 10 h between
Daphnia homogenate and control treatment, and this difference further
increased until 50 h after start. A significant difference in aggregation
behaviour between treatments was already present after 2 h, but the time lapse
between production and use of homogenates was not stated. From the above, it
may be concluded that the presence of alarm signals has previously been
proposed, but their basic properties and mode of action seem not to have been
fully appreciated and accounted for.
Specificity and distribution of signals
In the present study, both D. galeata and D. pulex
induced morphological changes in D. galeata when used as feed for the
purpose of labelling predators. Similar results were found when extracts were
prepared from homogenates of the two species. However, this result does not
necessarily mean that the alarm signals produced by the two species represent
identical chemical compounds. It is still possible that the responses obtained
result from functional overlap of species-specific signals in closely related
species. Such signal overlaps are known to exist among cyprinid fishes
(Schutz, 1956;
Pfeiffer, 1962) and have also
been demonstrated in snails (Stenzler and
Atema, 1977). Accordingly, experience from other taxonomic groups
gives reason to believe that common signal features are present among related
species, but the apparent similarity in signal function between
Daphnia species does not rule out species specificity in signal
properties. However, no definite conclusions can be drawn from the current
data on differences in signal properties between Daphnia spp. and a
final answer to this problem must await more detailed functional and chemical
investigations.
It appears also that the inactive precursors may adopt different alarm
signal properties following passage through the gut of different predator
species. Evidence for such signal specificity in Daphnia has emerged
from data presented by other investigators. Dodson
(Dodson, 1989) measured the
predator-induced morphological responses of three common predators (phantom
midge larva, C. americanus; adult backswimmer, Notonecta
undulata; sunfish, Lepomis macrochirus) in seven species of
Daphnia. Each Daphnia species responded with
predator-specific morphological changes. Some of these Daphnia
species could also distinguish different predator stimuli by opposing
behavioural responses, i.e. sinking or rising in the water column
(Dodson, 1988). Support to
this finding was given by Loose et al.
(Loose et al., 1993),
who found it unlikely that fish and Chaoborus release identical
chemical cues and by Stibor and Lüning
(Stibor and Lüning, 1994)
who demonstrated that chemical cues from fish and invertebrate predators
influence life-history traits of Daphnia differently. It is common
textbook knowledge that the microbial life of the gut varies between animal
species, both between phyla as well as between species occupying different
ecological niches. Various strains of bacteria may possess enzyme systems
specific for their kind and could, accordingly, produce different alarm
signals from a common chemical precursor. Such a mechanism would explain how
taxonomically different predators might possess signal properties specific to
their species. If this proves to be true, our work may represent a fascinating
new gateway into the world of chemical communication.
Acknowledgments
We are indebted to two anonymous referees, who gave valuable input to improve the manuscript.
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Accepted December 10, 2002
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