Journal of Chemical Ecology, Vol. 20, No. 7, 1994
E C D Y S T E R O I D S F R O M Pycnogonum litorale ( A R T H R O P O D A , P A N T O P O D A ) A C T AS C H E M I C A L D E F E N S E A G A I N S T Carcinus maenas (CRUSTACEA, DECAPODA)
K.-H. TOMASCHKO Abteilung Allgemeine Zoologie, Universitiit Ulm Albert-Einstein-Allee 11, 89069 Ulm, Germany (Received September 28, 1993; accepted January 21, 1994)
Abstract--Pycnogonum litorale (Strfm) is unpalatable to the common shore crab Carcinus maenas, a generalist predator in the pycnogonid's habitat. A feeding bioassay reveals that the crabs are deterred by ecdysteroids that occur in high levels in all developmental stages of P. litorale. The total ecdysteroids in the pycnogonids reach 5.9 x 10 -4 M. The 20-hydroxyecdysone 22-acetate (20E22A), which is the predominant ecdysteroid in the pycnogonids, and 20hydroxyecdysone (20E), the arthropod molting hormone, were tested for their antifeedant effect on C. maenas. When contained in food pellets in homogeneous concentrations, 20E and 20E22A significantly reduced food consumption at 1.25 × 10 -4 and 5.0 x 10 -a molar levels, respectively. The present results demonstrate for the first time chemical defense in arthropods in a marine predator-prey relationship. Furthermore, they provide evidence that ES contained in one animal can act as feeding deterrents on another animal.
Key Words--Pycnogonids, Pycnogonum litorale, Pantopoda, Crustacea, Carcinus maenas, ecdysteroids, 20-hydroxyecdysone, 20-hydroxyecdysone 22-acetate, chemical defense, predator-prey interactions.
INTRODUCTION
Chemical feeding deterrents have been isolated from a variety of terrestrial and freshwater atthropods (see reviews in Blum, 1981; Pasteels and Gr6goire, 1983; Dettner, 1987), whereas chemical defense in marine arthropods is largely uninvestigated. The present paper reports on the discovery of chemical defense in an archaic marine arthropod, Pycnogonum litorale. 1445 0098-0331/94/0700-1445507.00/0© 1994 Plenum Publishing Corporation
1446
TOmASCHKO
The pycnogonids (pantopods) are, apart from the xiphosurans, the only extant primitive marine chelicerates. Their fossil history can be traced back to the Devonian (Bergstr6m et al., 1980). P. litorale is unique in that it contains the highest ecdysteroid concentrations ever found in animals. In juvenile males the total ecdysteroid content is up to 0.1% of their body dry weight (Tomaschko and Bfickmann, 1993), corresponding to a 5.9 x 10 -4 molar concentration in living animals. Not only are the high levels of the ecdysteroids (ES) unusual, but are their chemical structures. Apart from 20-hydroxyecdysone (20E), the following seven ES have been found in all developmental stages of P. litorale (for chemical characterization see Brickmann et al., 1986): 20-hydroxyecdysone 22-glycolate, the 25 R and 25 S isomers of 20,26-dihydroxyecdysone 22-acetate, 22-deoxy-20,26-dihydroxyecdysone, 20-hydroxyecdysone 22-acetate (20E22A), 22-deoxy-20-hydroxyecdysone, and ecdysone 22-glycolate. The predominant compound is 20E22A. It has been found only during in vitro incubations in Drosophila melanogaster (Mar6y et al., 1988) and in a snail (Garcia et al., 1986). The other six ES are unique in the animal kingdom. One function of the ES in P. litorale, which is common to all arthropods, is the control of molting (Brickmann and Tomaschko, 1992). This, however, does not explain the presence of ES in nonmolting stages, i.e., in embryos and adults. Regardless of sex, ES concentrations are so high in all developmental stages that additional functions have been suspected (Tomaschko and Brickmann, 1993). An important hint at a possible allelochemical function of ES is contained in the results of Takahashi and Kittredge (1973). For seawater containing 20E in 1 0 - 6 - 1 0 - 9 molar concentrations, the normal feeding response in males of the lined shore crab, Pachygrapsus crassipes, is suppressed. This led to the idea that pycnogonid ES may serve as protection against predation by crustaceans. The assumption that P. litorale possesses some kind of chemical defense seemed reasonable, as all developmental stages, except the planktic first larvae, have very limited locomotor ability. The animals are found on wave-washed shores and live ectoparasitically in exposed positions on coelenterates or attached to hard substrates (Arnaud and Bamber, 1987). Having no physical defensive weapons, they would be easy prey for potential predators. In order to study the defensive properties of the pycnogonid ES, a feeding bioassay with the common shore crab Carcinus maenas has been developed. C. maenas is a widespread generalist predator in the northeast Atlantic littoral (Crothers, 1968), easily capable of coping with prey of the size of P. litorale. It reaches high abundance in the interstices of rocky shores and jetties, where it is often closely associated with P. litorale (K.-H. Tomaschko, personal observation). In a first experiment, living pycnogonids were offered to C. maenas in order to find out whether they are eaten or rejected by the crabs. Next, the
ECDYSTEROIDS AS CHEMICAL DEFENSE
1447
inhibition of crab feeding by lyophilized and powderized pycnogonids was tested. Finally, the feeding deterrent properties of 20E and 20E22A were investigated by adding the ES in different amounts to standardized food pellets. 20E22A is the most abundant ES in P. litorale, representing between 66.2% and 85.5% of the total ES in all developmental stages (Tomaschko and Biickmann, 1993). 20E makes up only a few percent of the pycnogonid ES, yet it is of interest because it represents the active form of the molting hormone in both C. maenas and P. litorale.
METHODS AND MATERIALS
Preparation of Experimental Diet. The semiartificial basic diet for 6". maenas consisted of water, gelatin, and, as a flavoring agent, lyophilized and ground mantle tissue of Mytilus edulis (20 : 2 : 1; w/w/w). Ingredients were stirred at 90°C for 5 min, in order to dissolve the gelatin and disperse the mussel powder. Pellets were prepared by pipetting 15-/A portions of the mixture into holes (2.5 mm diameter) in a Plexiglas plate (3 mm thick). Pellets congealed within a few minutes and could be easily removed. Pycnogonid powder was obtained by lyophilizing 30 adult females and thoroughly grinding them in a mortar. The powder was mixed with the basic diet at 38°C for 5 min at concentrations of 40%, 20%, 10%, and 5% (w/w), respectively. ES-treated pellets were prepared by mixing the basic diet with 20E (Sigma) and 20E22A (Sigma), respectively. From 2 × 10 -3 M stock solutions, lower concentrations were obtained by adding adequate amounts of the basic diet and mixing it for 5 min at 38°C. Carcinus Feeding Bioassay. Following 24 hr of starvation, adult male and female crabs were placed individually in glass aquaria (25 x 15 × 10 cm) containing 500 ml artificial seawater, where they were randomly assigned to treatment. Control pellets (basic diet) and treated pellets were presented singly and altemately to the individual crabs. Initial responses of the crabs after pellet presentation were scored as either 100% acceptance (pellet swallowed completely), partial acceptance (part of the pellet swallowed, estimated in percent), or rejection (pellet first mouthed and then completely rejected, 0% acceptance). In the case of partial or complete rejection, the pellet was immediately removed from the aquarium in order to avoid repeated mouthing, which otherwise occurred at a rate of approximately 20%. Water was renewed after each offering of treated pellets. The number of pellets for each crab was restricted to 20 per day. Crabs ignoring a control pellet were regarded as saturated and not taken into account for that day. Significances were calculated with the Wilcoxon, Mann, and Whitney test. Biological Material. P. litorale was reared as described previously (Brick-
1448
TOMASCHKO
mann and Tomaschko, 1992). Adult males and females of C. maenas had a carapace width of 30--40 mm and were collected near Wilhelmshaven, North Sea, Germany, at low tide. They were reared in a 400 liter tank, each animal isolated in a cubic container (10 × 10 x 10 cm) in artificial seawater (Tropic Matin) at a salinity of 2.7% and a constant temperature of 15°C in a 12L: 12D photoperiod. Every other day the crabs were fed to saturation with mantle tissue of Mytilus edulis.
RESULTS
Feeding Experiments with Intact Pycnogonids. Intact, living adult pycnogonids (25 males and 25 females) were each offered to an individually caged C. maenas, 16 pycnogonids (nine males and seven females) were not attacked (Figure 1). In response to the presentation of the pycnogonids, the crabs intensified ventilation movements of their mouth parts for 10-60 sec. However, they did not touch the pycnogonids and eventually moved away. Twenty-nine pycnogonids (14 males and 15 females) were released unharmed after having been briefly seized with the chelipeds and palpated with the mouth parts for about 5-30 sec. Five pycnogonids (two males and three females) were released after suffering injuries from intensive mouthing for 5-30 sec by the crabs' mandibles. The cuticle of a walking leg was punctured and body fluid leaked out. However, in no case were discernible parts of the pycnogonids ingested. All pycnogonids were still alive 30 days after the experiment. It is obvious that P. litorale is not consumed by C. maenas. Predator Deterrence of Pycnogonid Powder. This experiment was performed in order to exclude the possibility that the nonacceptance by C. maenas 15 14 13
12 "~ u c "6
----
--
11 10 9 8
----
7
--
_ 3
0
--
Living Pycnogonids
m
1 Ignored
II
. ,es females
mouthed
mouthed
and released
and released
unharmed
injured
Flc. l. Fate of 50 adult pycnogonids offered to C.
moenas.
ECDYSTEROIDS AS
CHEMICAL
1449
DEFENSE
could be due to the behavior of living pycnogonids and/or to the physical protection by their thick cuticles. Lyophilization of 30 adult female pycnogonids reduced their weight (3.1 g) to 29% (0.9 g). Food pellets containing 40% (w/ w) pycnogonid powder were completely rejected by all crabs. At the 30%, 20%, 10 %, and 5 % levels, the food consumption was inhibited significantly by 92.3 %, 87.2%, 59.1%, and 19.3%, respectively (Figure 2). These results clearly show that the feeding deterrency for the crab is most likely due to chemical constituents in P. litorale. Predator Deterrence o f Pycnogonid Ecdysteroids. Both 20E and 20E22A dose-dependently reduced artificial food acceptance by C. maenas. There was no significant difference in food acceptance between male and female crabs. Control pellets as well as all ES-treated pellets were seized by the crabs with their chelipeds and carried to the mouth. All control pellets were ingested within 5-10 sec. ES-treated pellets were, in case of rejection, either released intact after a few seconds or lacerated and cast out by ventilatory movements of the exopodites within 10-30 sec. Even in cases of acceptance, the ecdysteroidtreated pellets were often mouthed for 30-60 sec before being partially or completely ingested. At a 20.0 x 10 -4 molar level, 20E completely inhibited artificial food consumption (Figure 3, for natural concentrations see Discussion). All pellets
100
--
90
--
n = 26
0.025
80-.c
8 nO 0 0 LL
70
--
60
--
50
--
40
--
30
--
20
--
10
--
< p < 0.05
U n=22 p < 0.005
Lyophilized Pycnogonids
I
m
n.2 4.
p < 0.005
0 0
5
10
20
n = 25 p
E C D Y S T E R O I D S F R O M Pycnogonum litorale ( A R T H R O P O D A , P A N T O P O D A ) A C T AS C H E M I C A L D E F E N S E A G A I N S T Carcinus maenas (CRUSTACEA, DECAPODA)
K.-H. TOMASCHKO Abteilung Allgemeine Zoologie, Universitiit Ulm Albert-Einstein-Allee 11, 89069 Ulm, Germany (Received September 28, 1993; accepted January 21, 1994)
Abstract--Pycnogonum litorale (Strfm) is unpalatable to the common shore crab Carcinus maenas, a generalist predator in the pycnogonid's habitat. A feeding bioassay reveals that the crabs are deterred by ecdysteroids that occur in high levels in all developmental stages of P. litorale. The total ecdysteroids in the pycnogonids reach 5.9 x 10 -4 M. The 20-hydroxyecdysone 22-acetate (20E22A), which is the predominant ecdysteroid in the pycnogonids, and 20hydroxyecdysone (20E), the arthropod molting hormone, were tested for their antifeedant effect on C. maenas. When contained in food pellets in homogeneous concentrations, 20E and 20E22A significantly reduced food consumption at 1.25 × 10 -4 and 5.0 x 10 -a molar levels, respectively. The present results demonstrate for the first time chemical defense in arthropods in a marine predator-prey relationship. Furthermore, they provide evidence that ES contained in one animal can act as feeding deterrents on another animal.
Key Words--Pycnogonids, Pycnogonum litorale, Pantopoda, Crustacea, Carcinus maenas, ecdysteroids, 20-hydroxyecdysone, 20-hydroxyecdysone 22-acetate, chemical defense, predator-prey interactions.
INTRODUCTION
Chemical feeding deterrents have been isolated from a variety of terrestrial and freshwater atthropods (see reviews in Blum, 1981; Pasteels and Gr6goire, 1983; Dettner, 1987), whereas chemical defense in marine arthropods is largely uninvestigated. The present paper reports on the discovery of chemical defense in an archaic marine arthropod, Pycnogonum litorale. 1445 0098-0331/94/0700-1445507.00/0© 1994 Plenum Publishing Corporation
1446
TOmASCHKO
The pycnogonids (pantopods) are, apart from the xiphosurans, the only extant primitive marine chelicerates. Their fossil history can be traced back to the Devonian (Bergstr6m et al., 1980). P. litorale is unique in that it contains the highest ecdysteroid concentrations ever found in animals. In juvenile males the total ecdysteroid content is up to 0.1% of their body dry weight (Tomaschko and Bfickmann, 1993), corresponding to a 5.9 x 10 -4 molar concentration in living animals. Not only are the high levels of the ecdysteroids (ES) unusual, but are their chemical structures. Apart from 20-hydroxyecdysone (20E), the following seven ES have been found in all developmental stages of P. litorale (for chemical characterization see Brickmann et al., 1986): 20-hydroxyecdysone 22-glycolate, the 25 R and 25 S isomers of 20,26-dihydroxyecdysone 22-acetate, 22-deoxy-20,26-dihydroxyecdysone, 20-hydroxyecdysone 22-acetate (20E22A), 22-deoxy-20-hydroxyecdysone, and ecdysone 22-glycolate. The predominant compound is 20E22A. It has been found only during in vitro incubations in Drosophila melanogaster (Mar6y et al., 1988) and in a snail (Garcia et al., 1986). The other six ES are unique in the animal kingdom. One function of the ES in P. litorale, which is common to all arthropods, is the control of molting (Brickmann and Tomaschko, 1992). This, however, does not explain the presence of ES in nonmolting stages, i.e., in embryos and adults. Regardless of sex, ES concentrations are so high in all developmental stages that additional functions have been suspected (Tomaschko and Brickmann, 1993). An important hint at a possible allelochemical function of ES is contained in the results of Takahashi and Kittredge (1973). For seawater containing 20E in 1 0 - 6 - 1 0 - 9 molar concentrations, the normal feeding response in males of the lined shore crab, Pachygrapsus crassipes, is suppressed. This led to the idea that pycnogonid ES may serve as protection against predation by crustaceans. The assumption that P. litorale possesses some kind of chemical defense seemed reasonable, as all developmental stages, except the planktic first larvae, have very limited locomotor ability. The animals are found on wave-washed shores and live ectoparasitically in exposed positions on coelenterates or attached to hard substrates (Arnaud and Bamber, 1987). Having no physical defensive weapons, they would be easy prey for potential predators. In order to study the defensive properties of the pycnogonid ES, a feeding bioassay with the common shore crab Carcinus maenas has been developed. C. maenas is a widespread generalist predator in the northeast Atlantic littoral (Crothers, 1968), easily capable of coping with prey of the size of P. litorale. It reaches high abundance in the interstices of rocky shores and jetties, where it is often closely associated with P. litorale (K.-H. Tomaschko, personal observation). In a first experiment, living pycnogonids were offered to C. maenas in order to find out whether they are eaten or rejected by the crabs. Next, the
ECDYSTEROIDS AS CHEMICAL DEFENSE
1447
inhibition of crab feeding by lyophilized and powderized pycnogonids was tested. Finally, the feeding deterrent properties of 20E and 20E22A were investigated by adding the ES in different amounts to standardized food pellets. 20E22A is the most abundant ES in P. litorale, representing between 66.2% and 85.5% of the total ES in all developmental stages (Tomaschko and Biickmann, 1993). 20E makes up only a few percent of the pycnogonid ES, yet it is of interest because it represents the active form of the molting hormone in both C. maenas and P. litorale.
METHODS AND MATERIALS
Preparation of Experimental Diet. The semiartificial basic diet for 6". maenas consisted of water, gelatin, and, as a flavoring agent, lyophilized and ground mantle tissue of Mytilus edulis (20 : 2 : 1; w/w/w). Ingredients were stirred at 90°C for 5 min, in order to dissolve the gelatin and disperse the mussel powder. Pellets were prepared by pipetting 15-/A portions of the mixture into holes (2.5 mm diameter) in a Plexiglas plate (3 mm thick). Pellets congealed within a few minutes and could be easily removed. Pycnogonid powder was obtained by lyophilizing 30 adult females and thoroughly grinding them in a mortar. The powder was mixed with the basic diet at 38°C for 5 min at concentrations of 40%, 20%, 10%, and 5% (w/w), respectively. ES-treated pellets were prepared by mixing the basic diet with 20E (Sigma) and 20E22A (Sigma), respectively. From 2 × 10 -3 M stock solutions, lower concentrations were obtained by adding adequate amounts of the basic diet and mixing it for 5 min at 38°C. Carcinus Feeding Bioassay. Following 24 hr of starvation, adult male and female crabs were placed individually in glass aquaria (25 x 15 × 10 cm) containing 500 ml artificial seawater, where they were randomly assigned to treatment. Control pellets (basic diet) and treated pellets were presented singly and altemately to the individual crabs. Initial responses of the crabs after pellet presentation were scored as either 100% acceptance (pellet swallowed completely), partial acceptance (part of the pellet swallowed, estimated in percent), or rejection (pellet first mouthed and then completely rejected, 0% acceptance). In the case of partial or complete rejection, the pellet was immediately removed from the aquarium in order to avoid repeated mouthing, which otherwise occurred at a rate of approximately 20%. Water was renewed after each offering of treated pellets. The number of pellets for each crab was restricted to 20 per day. Crabs ignoring a control pellet were regarded as saturated and not taken into account for that day. Significances were calculated with the Wilcoxon, Mann, and Whitney test. Biological Material. P. litorale was reared as described previously (Brick-
1448
TOMASCHKO
mann and Tomaschko, 1992). Adult males and females of C. maenas had a carapace width of 30--40 mm and were collected near Wilhelmshaven, North Sea, Germany, at low tide. They were reared in a 400 liter tank, each animal isolated in a cubic container (10 × 10 x 10 cm) in artificial seawater (Tropic Matin) at a salinity of 2.7% and a constant temperature of 15°C in a 12L: 12D photoperiod. Every other day the crabs were fed to saturation with mantle tissue of Mytilus edulis.
RESULTS
Feeding Experiments with Intact Pycnogonids. Intact, living adult pycnogonids (25 males and 25 females) were each offered to an individually caged C. maenas, 16 pycnogonids (nine males and seven females) were not attacked (Figure 1). In response to the presentation of the pycnogonids, the crabs intensified ventilation movements of their mouth parts for 10-60 sec. However, they did not touch the pycnogonids and eventually moved away. Twenty-nine pycnogonids (14 males and 15 females) were released unharmed after having been briefly seized with the chelipeds and palpated with the mouth parts for about 5-30 sec. Five pycnogonids (two males and three females) were released after suffering injuries from intensive mouthing for 5-30 sec by the crabs' mandibles. The cuticle of a walking leg was punctured and body fluid leaked out. However, in no case were discernible parts of the pycnogonids ingested. All pycnogonids were still alive 30 days after the experiment. It is obvious that P. litorale is not consumed by C. maenas. Predator Deterrence of Pycnogonid Powder. This experiment was performed in order to exclude the possibility that the nonacceptance by C. maenas 15 14 13
12 "~ u c "6
----
--
11 10 9 8
----
7
--
_ 3
0
--
Living Pycnogonids
m
1 Ignored
II
. ,es females
mouthed
mouthed
and released
and released
unharmed
injured
Flc. l. Fate of 50 adult pycnogonids offered to C.
moenas.
ECDYSTEROIDS AS
CHEMICAL
1449
DEFENSE
could be due to the behavior of living pycnogonids and/or to the physical protection by their thick cuticles. Lyophilization of 30 adult female pycnogonids reduced their weight (3.1 g) to 29% (0.9 g). Food pellets containing 40% (w/ w) pycnogonid powder were completely rejected by all crabs. At the 30%, 20%, 10 %, and 5 % levels, the food consumption was inhibited significantly by 92.3 %, 87.2%, 59.1%, and 19.3%, respectively (Figure 2). These results clearly show that the feeding deterrency for the crab is most likely due to chemical constituents in P. litorale. Predator Deterrence o f Pycnogonid Ecdysteroids. Both 20E and 20E22A dose-dependently reduced artificial food acceptance by C. maenas. There was no significant difference in food acceptance between male and female crabs. Control pellets as well as all ES-treated pellets were seized by the crabs with their chelipeds and carried to the mouth. All control pellets were ingested within 5-10 sec. ES-treated pellets were, in case of rejection, either released intact after a few seconds or lacerated and cast out by ventilatory movements of the exopodites within 10-30 sec. Even in cases of acceptance, the ecdysteroidtreated pellets were often mouthed for 30-60 sec before being partially or completely ingested. At a 20.0 x 10 -4 molar level, 20E completely inhibited artificial food consumption (Figure 3, for natural concentrations see Discussion). All pellets
100
--
90
--
n = 26
0.025
80-.c
8 nO 0 0 LL
70
--
60
--
50
--
40
--
30
--
20
--
10
--
< p < 0.05
U n=22 p < 0.005
Lyophilized Pycnogonids
I
m
n.2 4.
p < 0.005
0 0
5
10
20
n = 25 p
