- Email: [email protected]
Metabolic and behavioral alterations in the crab Hemigrapsus crenulatus (Milne-Edwards 1837) induced by its acanthocephalan parasite Profilicollis antarcticus (Zdzitowiecki 1985)
Journal of Experimental Marine Biology and Ecology, 228 (1998) 73–82
L
Metabolic and behavioral alterations in the crab Hemigrapsus crenulatus (Milne-Edwards 1837) induced by its acanthocephalan parasite Profilicollis antarcticus (Zdzitowiecki 1985) Pilar A. Haye, F. Patricio Ojeda* ´ , P. Universidad Catolica ´ de Chile, Casilla 114 -D, Santiago, Chile Departamento de Ecologıa Received 8 August 1997; received in revised form 3 November 1997; accepted 19 December 1997
Abstract Acanthocephalans are parasites with complex life cycles. The parasitic larval stages are found mostly within crustaceans, while the adults live in the digestive tract of vertebrates that prey upon the intermediate hosts. Transition of the cystacanth larvae to the definitive host is mediated by a trophic interaction. To ensure transmission to the final vertebrate host, these parasites are known to induce behavioral changes in the intermediate host, increasing its vulnerability to predation. The acanthocephalan, Profilicollis antarcticus (Zdzitowiecki), has as intermediate host the estuarine crab, Hemigrapsus crenulatus (Milne-Edwards), and as definitive host, the gull, Larus dominicanus. We hypothesized that persistent behavioral alterations observed in crabs infected with acanthocephalans are determined by physiological changes, which are expressed in alterations of the metabolic rate of the intermediate host. Determinations of oxygen consumption were carried out for parasitized and control (non-parasitized) crabs, and metabolic rates were calculated based on oxygen consumption. The crabs infected with cystacanths of P. antarcticus had higher metabolic rates than the control crabs. Patterns of activity of the parasitized and control crabs were recorded according to seven behavioral displays arbitrarily defined. Parasitized crabs were much more active—and excited—than control crabs. These results suggest that the cystacanth larvae of the acanthocephalan, P. antarcticus, induces a phenotypic change in the crab, H. crenulatus, both in the metabolic rate and in activity patterns. This study represents the first experimental demonstration that altered behavior induced by acanthocephalan parasites on their hosts has a physiological basis. The physiological manipulation of the parasite alters the metabolic rate of its host, which is expressed in behavioral changes that increase its vulnerability to predators which are the definitive hosts of the parasite. 1998 Elsevier Science B.V. All rights reserved. Keywords: Parasite–host relationship; Parasite transmission; Acanthocephalans; Behavioral changes
*Corresponding author: Tel.: 562 6862729; fax: 562 2222621; e-mail: [email protected] 0022-0981 / 98 / $ – see front matter 1998 Elsevier Science B.V. All rights reserved. PII: S0022-0981( 98 )00007-0
74
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
1. Introduction Some hosts of helminth parasites show altered phenotypic characteristics following infestation. The most frequently reported are changes in pigmentation of the carapace and in behavior (see Stamp, 1981; Webber et al., 1987a,b; Dobson, 1988; Lo Bue and Bell, 1993; Lafferty and Morris, 1996, among others). In infected intermediate hosts of parasites with complex life cycles (more than one host species in the life cycle) several behavioral alterations have been reported, the majority of which can be viewed as phenotypic changes that enhance the transmission of the parasite to the definitive host (Camp and Huizinga, 1979; Oetinger and Nickol, 1981, 1982; Moore, 1983, 1984a,b, 1987, 1995; Moore and Lasswell, 1986; Carmichael and Moore, 1991; Combes, 1991; Gotelli and Moore, 1992; Moore and Gotelli, 1992; Zachary et al., 1992; Carmichael et al., 1993; Hechtel et al., 1993). Larval stages of acanthocephalans are parasitic within crustaceans and insects. Adult stages are found in the digestive tracts of vertebrates, especially fish and birds, which live associated to water sources where they feed upon the intermediate hosts. When parasitic transmission depends on predation, it has often been suggested that the parasite may alter the host behavior, increasing the vulnerability of intermediate hosts to predation by the final host (Holmes and Bethel, 1972). The phenotypic changes that parasites induce on their intermediate hosts have been considered as alterations that have, as the ultimate goal, an increase in the parasite fitness by ensuring its transmission to the definitive host and thus allowing the development of the adult stage (Moore, 1983, 1984a,b; Webber et al., 1987a,b; Dobson, 1988; Hechtel et al., 1993; Lafferty and Morris, 1996). Nevertheless, there are alternative explanations for the phenotypic changes: it may well be an adaptation of the host (Stamp, 1981; Poulin, 1994), or it may be a byproduct of phylogenetic inertia of the parasite or of the host (i.e., that it was an adaptation in the common ancestor of the group, and that now is only a characteristic of the group and not a specific adaptation of the animal to its environment) (Moore and Gotelli, 1990, 1996). Thus, phenotypic alterations may only be a consequence of the infection without being adaptive for the parasite or for the host. The broadly accepted explanation, however, is that it is an adaptation of the host, because parasitism by definition is detrimental to the host, diminishing its fitness (Holmes and Zohar, 1990). The physiology of an organism is closely related to its behavior. Although this relationship may not be direct, the behavioral changes must have a physiological background (Thompson and Kavaliers, 1994). Parasitism generally results in physiological responses by the host, which are usually manifested in an altered behavior (Thompson and Kavaliers, 1994). Even though the nature of the phenotypic changes in infected hosts is unclear, some information exists on the biochemical and physiological mechanisms that underlie them. Understanding such mechanisms (that later lead to behavioral, morphological and other phenotypic changes) may help answer the question about the nature of the alterations. Many parasites that induce phenotypic changes are located in the muscle or nervous tissue of the host, causing systemic changes directly in the organs. The cystacanth larvae of the acanthocephalans are located in the hemocoelomic cavity of crabs, suggesting that the changes that parasites produce in their hosts may be biochemically induced through
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
75
the discharge of a substance to the coelom of the host (Moore, 1984b). Some neurohormones such as serotonin, dopamine, and octopamine are known to produce multiple effects in crustaceans, affecting the phenotype, the motor, ventilatory and cardiac activity, as well as coloration patterns (Livingstone et al., 1980; Beltz and Kravitz, 1986; Flamm and Harris-Warrick, 1986; Kravitz, 1988; Dickinson and Marder, 1989; Rajashekhar and Wilkens, 1991). These neurohormones are probably the compounds that mediate the alterations provoked by the parasites. There is also evidence of the existence of opioid systems on invertebrates that have functional roles (Thompson and Kavaliers, 1994). Because amines produce direct effects on the nervous system of crustaceans (either inhibiting or activating), it is likely that changes in some amines or other substances with endocrine effects may produce changes in the metabolic rate of infected hosts. Further, these changes in metabolic rates lead to behavioral changes. On the basis of this background, we hypothesized that behavioral alterations observed in intermediate hosts infected with acanthocephalans are determined by physiological changes, which are expressed in alterations of the metabolic rate of the intermediate host. The acanthocephalan, Profilicollis antarcticus (Zdzitowiecki), has as its intermediate host the estuarine crab Hemigrapsus crenulatus (Milne-Edwards), and as its definitive host the gull, Larus dominicanus. A trophic relationship mediates the transfer of the acanthocephalan from the larval cystacanth stage in the crab to the adult stage in the gull (Moore, 1987; Lozano, 1991; Pulgar et al., 1995). This trophic relation may be enhanced by a parasite- induced behavioral change on the crab. The main goal of this study was to determine whether the parasite, P. antarcticus, induces metabolic (oxygen consumption) and behavioral alterations (activity patterns) on the crab, H. crenulatus.
2. Materials and methods Crabs were manually collected in the Lenga estuary (36845’ S; 73810’ W) in March 1996 and transported to the laboratory in Santiago, where they were acclimatized in aerated aquaria for 30 days at 16.58C, an estuarine type salinity of 20 ppm, under a 12 h day–12 h night cycle, and feeding every 4–5 days.
2.1. Oxygen consumption Respiration rates of crabs were measured during daylight, all at the same hour, after 5 days without food to avoid any cost associated with digestion or biosynthesis of macromolecules (i.e., specific dynamic action). The specimens were introduced individually into a 520 ml airtight chamber. The chamber was equipped with a platinum oxygen microelectrode (Strathkelvin model 1302) connected to a Strathkelvin oxygen meter (model 781). Temperature was kept constant at 16.58C with a thermoregulated bath. Each individual was acclimated to the respirometry chamber for 1 h with fresh seawater. At the onset of the determination there was an input of fresh seawater (previously oxygenated to 100% saturation). Oxygen consumption was measured for 30–45 min, making sure that partial oxygen pressure would not drop below 70% of the
76
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
initial value. The oxidation of the chlorine of the electrode (that produces current) is proportional to the amount of oxygen in the water, which is recorded by the oxygen meter (previously calibrated for the salinity and temperature of the measurements, which were the same as in the acclimation conditions). The oxygen meter yields a percentage of oxygen in reference to the initial 100%. The amount of oxygen in solution in water depends on its salinity and temperature (at 20 ppm of salinity and 16.58C of temperature the water has 565 mg of oxygen / l, equivalent to 6.3 ml of oxygen / l). Two determinations of oxygen consumption, separated by a period of water renewal, were effected for each individual. After the second determination, the specimen was removed and its body mass (W ) was determined to the nearest g. Then a control was run in the absence of the animal. Oxygen consumption was expressed in mg O 2 consumed per min, calculated from the percentage decrease in partial pressure of oxygen, water volume present in the chamber, and time in min. During the measurements we recorded every 3 min the value yielded by the oxygen meter of the percentage of oxygen in the metabolic chamber, and, finally, using linear regression we obtained the slope corresponding to the rate of oxygen decrease in the chamber. From the mean of the two determinations of oxygen consumption obtained for each individual, we subtracted the consumption of each control (chamber without crab). Oxygen consumption was expressed with reference to each individual’s wet weight. Throughout the determinations of oxygen consumption, all specimens of H. crenulatus remained inactive (in resting position) inside the chamber, hence these oxygen consumption measurements likely correspond most closely to the standard metabolic rate (Brett and Groves, 1979). Crabs were randomly chosen from the aquaria. After the blind metabolic determinations (i.e., it was not known a priori whether the crabs where parasitized or not), crabs were dissected to verify the presence of P. antarcticus cystacanths on the hemocoel, yielding 11 parasitized crabs and 11 control crabs (without parasites). Most infected crabs were parasitized by two cystacanths.
2.2. Behavioral observations To check for differences in activity patterns of parasitized and control crabs, video recordings were run for 20 min (using a Canon L1 video camera equipped with a 15 3 zoom lens) in 20 3 10 3 10 cm aquariums filled with 2 cm of water. All recordings were done under the same experimental conditions. After the videotaping, the crabs were dissected to determine the presence of cystacanths in the hemocoel (i.e., it was not known if a crab was parasitized when scoring its behavior), resulting in 9 control crabs and 9 parasitized crabs. All videotapings were performed under the same experimental conditions. We recorded the percentage of time crabs spent in each of seven recognizable behavioral displays. Four of these displays were considered as ‘active’ (involved some kind of movement): (a) movement of the non-locomotory appendages (i.e., jaws, antennae), (b) movement of all the appendages but without displacement, (c) slow displacement, and (d) fast displacement. Three other displays were considered as ‘inactive’ (absence of movement): (a) relaxed with no appendages contracted and all the
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
77
Fig. 1. Seven recognizable behavioral displays of the crabs studied. Inactivity (no movement): (1) Relaxed with no contracted muscles in the locomotory appendages. (2) Semialert, with one or two locomotory appendages contracted, and with half of the body out of the water. (3) Alert, with all the muscles of the locomotory appendages contracted, and the body completely out of the water. Activity (any kind of movement): (4) Movement of non-locomotory appendages only. (5) Movement of all appendages but without displacement. (6) Slow displacement. (7) Fast displacement. Displays 3, 5 and 7 are considered as excitatory states.
body inside the water, (b) semialert with the first or two first anterior appendages contracted and half the body outside the water, and (c) alert, with all the appendages contracted and the cephalothorax outside the water (Fig. 1). Three of the displays were classified as excitatory: inactive but alert, movement of all the appendages without displacement, and fast displacement. The average time (obtained from two periods of 5 min each) that crabs were in each of the seven displays was used to determine differences between parasitized and control crabs. After the experiments, all crabs were dissected to determine the presence or absence of parasites in the hemocoel. The data on oxygen consumption and behavior were statistically analyzed using a Mann-Whitney nonparametric test (M-W) (Siegel and Castellan, 1988).
3. Results
3.1. Oxygen consumption Crabs infected with cystacanths had higher metabolic rates than control crabs (X62 SE: 0.12560.019, vs. 0.07960.014 mg O 2 l / min g, for infected and non-infected crabs, respectively; U 5 11.5, p 5 0.0013) (Fig. 2). Comparisons between the two groups indicated that their body masses were statistically similar (X62 SE: 8.71262.053; 9.12762.102, for infected and non-infected crabs, respectively; U 5 55.0, p 5 0.718).
78
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
Fig. 2. Oxygen consumption of parasitized and non-parasitized crabs expressed in mg oxygen ml / min g. Vertical lines represent 2 SE.
3.2. Behavior The parasitized crabs were significantly more active than the control crabs (80.6% vs. 35.5%; U 5 0.1 p 5 0.0003) (Fig. 3). Parasitized crabs spent more time moving all the appendages without displacement (29.2% of the total time in parasitized crabs vs. 2.2% of total time for controls; U 5 6.5, p 5 0.0027) and in fast displacement (34.2% of the total time in infected crabs, vs. 4.5% in control crabs; U 5 2; p 5 0.0007) (Fig. 4). In contrast, control crabs spent more time in resting position than parasitized ones (40.9% of the total time in control crabs vs. 5.1% in parasitized crabs; U 5 2, p 5 0.0007). For
Fig. 3. Percentage of time that control and parasitized crabs remained in activity. Vertical lines represent 2 SE.
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
79
Fig. 4. Percentage of time that control and parasitized crabs were in each of the seven recognizable behavioral displays previously described. Numbers in squares indicate excitatory attitudes. Vertical lines represent 2 SE.
the remainder of the positions or displays, there were no significant differences between both groups of crabs ( p . 0.05).
4. Discussion Thompson and Kavaliers (1994) proposed that in studies of parasitism it is possible to integrate various levels of biological organization, allowing links among behavior, physiology and ecology. Our study shows a close relationship between behavior and physiology, which supports our hypothesis that alterations of host behavior are correlated with changes in metabolic rate. Parasitized crabs had higher metabolic rates and altered activity patterns. Differences in the oxygen uptake may be due to several factors, such as type of diet, temperature, acclimation, hour of the day (circadian factors), oxygen availability, and body mass (Steffensen, 1989). The first five factors were controlled for and set constant for all crabs in the experiments. Moreover, parasitized crabs were not only more active than non-infected crabs, but also spent more time in two of the three excitatory attitudes. These excitatory attitudes, specifically movement of all appendages with or without displacement, may render crabs more visually exposed in the environment, increasing their vulnerability to predators. In Lenga estuary there are significantly higher numbers of infected crabs walking in the sand out of the water in comparison to the non-infected crabs, which usually remain on the bottom of the estuary (M. George-Nascimento, pers. comm.). This agrees well with our findings. Further, our results are consistent with the well documented hypothesis that parasites alter host behavior, which in turn may increase susceptibility of intermediate
80
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
host to predation by the final host (Holmes and Bethel, 1972; Lafferty and Morris, 1996). Visual cues are among the main stimuli for predators to detect prey. If the prey exhibits a coloration that in any way is more arresting to the predator, or if it spends more time moving about, then it may be more detectable by the predator. Profilicollis antarcticus seems to be capable of causing this type of phenotypic changes in H. crenulatus, which render the crabs more susceptible to predation and in that way the parasite may complete its development and life cycle in the new host (Pulgar et al., 1995). Pulgar et al. (1995) found that parasitized H. crenulatus presented a different pattern of carapace coloration in comparison to non-parasitized crabs. In the laboratory they measured several crab behaviors in relation to water (hydrotaxis), light (phototaxis) and presence of a simulated predator. They did not find significant differences between parasitized and control crabs. However, we think their results may be flawed for two reasons: First, the acclimation times used were very short (48 h). A period of 4 weeks is a more reasonable time for acclimation, to allow the organisms to acquire a new endocrine and nervous stable state (important for physiological and behavioral analyses). Second, crabs were artificially inoculated, so they should have done a sham inoculation (they missed a control group). Although in our study we did not test for behavioral differences correlated with special environmental stimuli (e.g., light, water), it may be expected, that parasitized crabs are more sentient than non-parasitized ones. The present study represents the first experimental demonstration that altered behaviors induced by acanthocephalan parasites in their hosts have a physiological background. Indeed, it is a physiological manipulation of the parasite by altering the metabolic rates of its host, which is expressed in behavioral changes that may increase its vulnerability to predators, which are the final hosts for the parasite.
Acknowledgements ´ We thank G. Fernandez for his constant help in keeping the oxygen consumption ´ system in good shape, and for always being there when we had problems, C.W. Caceres for his help on data analysis, and M. George-Nascimento, J. Pulgar and the rest of the members of George-Nascimento’s laboratory for the collection of crabs, and for their ˜ and S. Navarrete important comments on this work. F. Bozinovic, F. Jaksic, A. Munoz and two anonymous reviewers made helpful comments that greatly improved this manuscript. This study was funded by FONDECYT Grant 1930420.
References Beltz, B.S., Kravitz, E.A., 1986. Aminergic and peptidergic neuromodulation in Crustacea. Journal of Experimental Biology 124, 115–141. Brett, J.R., Groves, T.D., 1979. Physiological energetics. In: Hoar, W.S. (Ed.), Fish Physiology, Vol. VIII: Bioenegetics and Growth, Academic Press, New York, pp. 279–352.
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
81
Camp, J.W., Huizinga, H.W., 1979. Altered color, behavior and predation susceptibility of the isopod Asellus intermedius infected with Acanthocephalus dirus. Journal of Parasitology 65, 667–669. Carmichael, L.M., Moore, J., 1991. A comparison of behavioral alterations in the brown cockroach, Periplaneta americana, infected with the acanthocephalan Moniliformis moniliformis. Journal of Parasitology 77, 931–936. Carmichael, L.M., Moore, J., Bjostad, L.B., 1993. Parasitism and decreased response to sex pheromones in male Periplaneta americana (Dictyoptera: Blattidae). Journal of Insect Behavior 6, 25–32. Combes, C., 1991. Ethological aspects of parasite transmission. American Naturalist 138, 866–880. Dickinson, P.S., Marder, E., 1989. Peptidergic modulation of a multioscillator system in the lobster. I. Activation of the cardiac sac motor pattern by the neuropeptides proctolin and red pigment-concentrating hormone. Journal of Neurophysiology 61, 833–844. Dobson, A.P., 1988. The population biology of parasite-induced changes in host behavior. The Quaterly Review of Biology 63, 139–165. Flamm, R.E., Harris-Warrick, R.M., 1986. Aminergic modulation in lobster stomatogastric ganglion. II. Target neurons of dopamine, octopamine, and serotonine within the pyloric circuit. The American Physiological Society 55, 866–881. Gotelli, N.J., Moore, J., 1992. Altered behavior in a cockroach–acanthocephalan association. Animal Behavior 43, 949–959. Hechtel, L.J., Johnson, C.L., Juliano, S.A., 1993. Modification of antipredator behavior of Caecidotea intermedius by its parasite Acanthocephalus dirus. Ecology 74, 710–713. Holmes, J.C., Bethel, W.B., 1972. Modification of intermediate host behaviour by parasites. Zoological Journal of the Linnean Society 51, 123–149. Holmes, J.C., Zohar, S., 1990. Pathology and host behaviour by parasites. In: Barnard, C.J., Behnke J.M. (Eds.), Parasitism and Host Behaviour, Taylor and Francis, New York, pp. 34–63. Kravitz, E.A., 1988. Hormonal control of behaviour: Amines and the biasing of behavioural output in lobster. Science 241, 1775–1781. Lafferty, K.D., Morris, A.K., 1996. Altered behavior of parasitized killfish increases susceptibility to predation by bird final hosts. Ecology 77, 1390–1397. Livingstone, M.S., Harris-Warrick, R.M., Kravitz, E.A., 1980. Serotonine and Octopamine produce opposite postures in lobsters. Science 208, 76–79. Lo Bue, C.P., Bell, M.A., 1993. Phenotypic manipulation by the cestode parasite Schistocephalus solidus of its intermediate host Gasterosteus aculeatus, the threespine stickleback. American Naturalist 142, 725–735. Lozano, G.A., 1991. Optimal foraging theory: A possible role for parasites. Oikos 60, 391–395. Moore, J., 1983. Responses of an avian predator and its isopod prey to an acanthocephalan parasite. Ecology 64, 1000–1015. Moore, J., 1984a. Altered behavioral responses in intermediate host—an acanthocephalan parasite strategy. American Naturalist 123, 572–577. Moore, J., 1984b. Parasites that change the behavior of their hosts. Scientific American 250, 108–115. Moore, J., 1987. Some roles of parasitic helminths in trophic interactions. A view from North America. Revista Chilena de Historia Natural 60, 159–179. Moore, J., 1995. The behavior of parasitized animals. BioScience 45, 89–96. Moore, J., Gotelli, N.J., 1990. A phylogenetic perspective on the evolution of altered host behaviours: A critical look at the manipulation hypothesis. In: Barnard, C.J., Behnke J.M. (Eds.), Parasitism and Host Behaviour, Taylor and Francis, New York, pp. 193–229. Moore, J., Gotelli, N.J., 1992. Moniliformis moniliformis increases cryptic behaviors in the cockroach Supella longipalpa. Journal of Parasitology 78, 49–53. Moore, J., Gotelli, N.J., 1996. Evolutionary patterns of altered behavior and susceptibility in parasitized hosts. Evolution 50, 807–819. Moore, J., Lasswell, J., 1986. Altered behavior in isopods (Armadillidium vulgare) infected with the nematode Dispharynx nasuta. Journal of Parasitology 72, 186–189. Oetinger, D.F., Nickol, B.B., 1981. Effects of acanthocephalans on pigmentation of freshwater isopods. Journal of Parasitology 67, 672–684. Oetinger, D.F., Nickol, B.B., 1982. Developmental relationships between acanthocephalans and altered pigmentation in freshwater isopods. Journal of Parasitology 68, 463–469.
82
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
Poulin, R., 1994. Parasite manipulation of host behaviour: Should hosts always lose?. Oikos 70, 479–484. Pulgar, J., Aldana, M., Vergara, E., George-Nascimento, M., 1995. La conducta de la jaiba estuarina ´ al parasitismo por el acantocefalo ´ Hemigrapsus crenulatus (Milne-Edwards 1837) en relacion Profilicollis antarcticus (Zdzitowiecki 1985) en el sur de Chile. Revista Chilena de Historia Natural 68, 439–450. Rajashekhar, K.P., Wilkens, J.L., 1991. Dopamine and nicotine, but not serotonine, modulate the crustacean ventilatory pattern generator. Journal of Neurobiology 29, 680–691. Siegel, S., Castellan, N.J., 1988. Nonparametric statistics for the behavioral sciences, 2nd ed., McGraw-Hill, New York. Steffensen, J.F., 1989. Some errors in respirometry of aquatic breathers: How to avoid and correct for them. Fish Physiology and Biochemistry 6, 49–50. Stamp, N., 1981. Behavior of parasitized aposematic caterpillars: Advantageous to the parasitoid or the host?. American Naturalist 118, 715–725. Thompson, S.N., Kavaliers, M., 1994. Physiological bases for parasite-induced alterations of host behaviour. Parasitology 109, 119–138. Webber, R.A., Rau, M.E., Lewis, D.J., 1987a. The effects of Plagiorchis noblei (Trematoda: Plagiorchiidae) metacercariae on the behavior of Aedes aegypti larvae. Canadian Journal of Zoology 65, 1340–1342. Webber, R.A., Rau, M.E., Lewis, D.J., 1987b. The effects of Plagiorchis noblei (Trematoda: Plagiorchiidae) metacercariae on the susceptibility of Aedes aegypti larvae to predation by guppies (Poecilia reticulata) and meadow voles (Microtus pennsylvanicus). Canadian Journal of Zoology 65, 2346–2348. Zachary, A., Moore, J., Gotelli, N.J., 1992. Moniliformis moniliformis infection has no effect on some behaviors of the cockroach Diploptera punctata. Journal of Parasitology 78, 524–526.
L
Metabolic and behavioral alterations in the crab Hemigrapsus crenulatus (Milne-Edwards 1837) induced by its acanthocephalan parasite Profilicollis antarcticus (Zdzitowiecki 1985) Pilar A. Haye, F. Patricio Ojeda* ´ , P. Universidad Catolica ´ de Chile, Casilla 114 -D, Santiago, Chile Departamento de Ecologıa Received 8 August 1997; received in revised form 3 November 1997; accepted 19 December 1997
Abstract Acanthocephalans are parasites with complex life cycles. The parasitic larval stages are found mostly within crustaceans, while the adults live in the digestive tract of vertebrates that prey upon the intermediate hosts. Transition of the cystacanth larvae to the definitive host is mediated by a trophic interaction. To ensure transmission to the final vertebrate host, these parasites are known to induce behavioral changes in the intermediate host, increasing its vulnerability to predation. The acanthocephalan, Profilicollis antarcticus (Zdzitowiecki), has as intermediate host the estuarine crab, Hemigrapsus crenulatus (Milne-Edwards), and as definitive host, the gull, Larus dominicanus. We hypothesized that persistent behavioral alterations observed in crabs infected with acanthocephalans are determined by physiological changes, which are expressed in alterations of the metabolic rate of the intermediate host. Determinations of oxygen consumption were carried out for parasitized and control (non-parasitized) crabs, and metabolic rates were calculated based on oxygen consumption. The crabs infected with cystacanths of P. antarcticus had higher metabolic rates than the control crabs. Patterns of activity of the parasitized and control crabs were recorded according to seven behavioral displays arbitrarily defined. Parasitized crabs were much more active—and excited—than control crabs. These results suggest that the cystacanth larvae of the acanthocephalan, P. antarcticus, induces a phenotypic change in the crab, H. crenulatus, both in the metabolic rate and in activity patterns. This study represents the first experimental demonstration that altered behavior induced by acanthocephalan parasites on their hosts has a physiological basis. The physiological manipulation of the parasite alters the metabolic rate of its host, which is expressed in behavioral changes that increase its vulnerability to predators which are the definitive hosts of the parasite. 1998 Elsevier Science B.V. All rights reserved. Keywords: Parasite–host relationship; Parasite transmission; Acanthocephalans; Behavioral changes
*Corresponding author: Tel.: 562 6862729; fax: 562 2222621; e-mail: [email protected] 0022-0981 / 98 / $ – see front matter 1998 Elsevier Science B.V. All rights reserved. PII: S0022-0981( 98 )00007-0
74
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
1. Introduction Some hosts of helminth parasites show altered phenotypic characteristics following infestation. The most frequently reported are changes in pigmentation of the carapace and in behavior (see Stamp, 1981; Webber et al., 1987a,b; Dobson, 1988; Lo Bue and Bell, 1993; Lafferty and Morris, 1996, among others). In infected intermediate hosts of parasites with complex life cycles (more than one host species in the life cycle) several behavioral alterations have been reported, the majority of which can be viewed as phenotypic changes that enhance the transmission of the parasite to the definitive host (Camp and Huizinga, 1979; Oetinger and Nickol, 1981, 1982; Moore, 1983, 1984a,b, 1987, 1995; Moore and Lasswell, 1986; Carmichael and Moore, 1991; Combes, 1991; Gotelli and Moore, 1992; Moore and Gotelli, 1992; Zachary et al., 1992; Carmichael et al., 1993; Hechtel et al., 1993). Larval stages of acanthocephalans are parasitic within crustaceans and insects. Adult stages are found in the digestive tracts of vertebrates, especially fish and birds, which live associated to water sources where they feed upon the intermediate hosts. When parasitic transmission depends on predation, it has often been suggested that the parasite may alter the host behavior, increasing the vulnerability of intermediate hosts to predation by the final host (Holmes and Bethel, 1972). The phenotypic changes that parasites induce on their intermediate hosts have been considered as alterations that have, as the ultimate goal, an increase in the parasite fitness by ensuring its transmission to the definitive host and thus allowing the development of the adult stage (Moore, 1983, 1984a,b; Webber et al., 1987a,b; Dobson, 1988; Hechtel et al., 1993; Lafferty and Morris, 1996). Nevertheless, there are alternative explanations for the phenotypic changes: it may well be an adaptation of the host (Stamp, 1981; Poulin, 1994), or it may be a byproduct of phylogenetic inertia of the parasite or of the host (i.e., that it was an adaptation in the common ancestor of the group, and that now is only a characteristic of the group and not a specific adaptation of the animal to its environment) (Moore and Gotelli, 1990, 1996). Thus, phenotypic alterations may only be a consequence of the infection without being adaptive for the parasite or for the host. The broadly accepted explanation, however, is that it is an adaptation of the host, because parasitism by definition is detrimental to the host, diminishing its fitness (Holmes and Zohar, 1990). The physiology of an organism is closely related to its behavior. Although this relationship may not be direct, the behavioral changes must have a physiological background (Thompson and Kavaliers, 1994). Parasitism generally results in physiological responses by the host, which are usually manifested in an altered behavior (Thompson and Kavaliers, 1994). Even though the nature of the phenotypic changes in infected hosts is unclear, some information exists on the biochemical and physiological mechanisms that underlie them. Understanding such mechanisms (that later lead to behavioral, morphological and other phenotypic changes) may help answer the question about the nature of the alterations. Many parasites that induce phenotypic changes are located in the muscle or nervous tissue of the host, causing systemic changes directly in the organs. The cystacanth larvae of the acanthocephalans are located in the hemocoelomic cavity of crabs, suggesting that the changes that parasites produce in their hosts may be biochemically induced through
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
75
the discharge of a substance to the coelom of the host (Moore, 1984b). Some neurohormones such as serotonin, dopamine, and octopamine are known to produce multiple effects in crustaceans, affecting the phenotype, the motor, ventilatory and cardiac activity, as well as coloration patterns (Livingstone et al., 1980; Beltz and Kravitz, 1986; Flamm and Harris-Warrick, 1986; Kravitz, 1988; Dickinson and Marder, 1989; Rajashekhar and Wilkens, 1991). These neurohormones are probably the compounds that mediate the alterations provoked by the parasites. There is also evidence of the existence of opioid systems on invertebrates that have functional roles (Thompson and Kavaliers, 1994). Because amines produce direct effects on the nervous system of crustaceans (either inhibiting or activating), it is likely that changes in some amines or other substances with endocrine effects may produce changes in the metabolic rate of infected hosts. Further, these changes in metabolic rates lead to behavioral changes. On the basis of this background, we hypothesized that behavioral alterations observed in intermediate hosts infected with acanthocephalans are determined by physiological changes, which are expressed in alterations of the metabolic rate of the intermediate host. The acanthocephalan, Profilicollis antarcticus (Zdzitowiecki), has as its intermediate host the estuarine crab Hemigrapsus crenulatus (Milne-Edwards), and as its definitive host the gull, Larus dominicanus. A trophic relationship mediates the transfer of the acanthocephalan from the larval cystacanth stage in the crab to the adult stage in the gull (Moore, 1987; Lozano, 1991; Pulgar et al., 1995). This trophic relation may be enhanced by a parasite- induced behavioral change on the crab. The main goal of this study was to determine whether the parasite, P. antarcticus, induces metabolic (oxygen consumption) and behavioral alterations (activity patterns) on the crab, H. crenulatus.
2. Materials and methods Crabs were manually collected in the Lenga estuary (36845’ S; 73810’ W) in March 1996 and transported to the laboratory in Santiago, where they were acclimatized in aerated aquaria for 30 days at 16.58C, an estuarine type salinity of 20 ppm, under a 12 h day–12 h night cycle, and feeding every 4–5 days.
2.1. Oxygen consumption Respiration rates of crabs were measured during daylight, all at the same hour, after 5 days without food to avoid any cost associated with digestion or biosynthesis of macromolecules (i.e., specific dynamic action). The specimens were introduced individually into a 520 ml airtight chamber. The chamber was equipped with a platinum oxygen microelectrode (Strathkelvin model 1302) connected to a Strathkelvin oxygen meter (model 781). Temperature was kept constant at 16.58C with a thermoregulated bath. Each individual was acclimated to the respirometry chamber for 1 h with fresh seawater. At the onset of the determination there was an input of fresh seawater (previously oxygenated to 100% saturation). Oxygen consumption was measured for 30–45 min, making sure that partial oxygen pressure would not drop below 70% of the
76
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
initial value. The oxidation of the chlorine of the electrode (that produces current) is proportional to the amount of oxygen in the water, which is recorded by the oxygen meter (previously calibrated for the salinity and temperature of the measurements, which were the same as in the acclimation conditions). The oxygen meter yields a percentage of oxygen in reference to the initial 100%. The amount of oxygen in solution in water depends on its salinity and temperature (at 20 ppm of salinity and 16.58C of temperature the water has 565 mg of oxygen / l, equivalent to 6.3 ml of oxygen / l). Two determinations of oxygen consumption, separated by a period of water renewal, were effected for each individual. After the second determination, the specimen was removed and its body mass (W ) was determined to the nearest g. Then a control was run in the absence of the animal. Oxygen consumption was expressed in mg O 2 consumed per min, calculated from the percentage decrease in partial pressure of oxygen, water volume present in the chamber, and time in min. During the measurements we recorded every 3 min the value yielded by the oxygen meter of the percentage of oxygen in the metabolic chamber, and, finally, using linear regression we obtained the slope corresponding to the rate of oxygen decrease in the chamber. From the mean of the two determinations of oxygen consumption obtained for each individual, we subtracted the consumption of each control (chamber without crab). Oxygen consumption was expressed with reference to each individual’s wet weight. Throughout the determinations of oxygen consumption, all specimens of H. crenulatus remained inactive (in resting position) inside the chamber, hence these oxygen consumption measurements likely correspond most closely to the standard metabolic rate (Brett and Groves, 1979). Crabs were randomly chosen from the aquaria. After the blind metabolic determinations (i.e., it was not known a priori whether the crabs where parasitized or not), crabs were dissected to verify the presence of P. antarcticus cystacanths on the hemocoel, yielding 11 parasitized crabs and 11 control crabs (without parasites). Most infected crabs were parasitized by two cystacanths.
2.2. Behavioral observations To check for differences in activity patterns of parasitized and control crabs, video recordings were run for 20 min (using a Canon L1 video camera equipped with a 15 3 zoom lens) in 20 3 10 3 10 cm aquariums filled with 2 cm of water. All recordings were done under the same experimental conditions. After the videotaping, the crabs were dissected to determine the presence of cystacanths in the hemocoel (i.e., it was not known if a crab was parasitized when scoring its behavior), resulting in 9 control crabs and 9 parasitized crabs. All videotapings were performed under the same experimental conditions. We recorded the percentage of time crabs spent in each of seven recognizable behavioral displays. Four of these displays were considered as ‘active’ (involved some kind of movement): (a) movement of the non-locomotory appendages (i.e., jaws, antennae), (b) movement of all the appendages but without displacement, (c) slow displacement, and (d) fast displacement. Three other displays were considered as ‘inactive’ (absence of movement): (a) relaxed with no appendages contracted and all the
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
77
Fig. 1. Seven recognizable behavioral displays of the crabs studied. Inactivity (no movement): (1) Relaxed with no contracted muscles in the locomotory appendages. (2) Semialert, with one or two locomotory appendages contracted, and with half of the body out of the water. (3) Alert, with all the muscles of the locomotory appendages contracted, and the body completely out of the water. Activity (any kind of movement): (4) Movement of non-locomotory appendages only. (5) Movement of all appendages but without displacement. (6) Slow displacement. (7) Fast displacement. Displays 3, 5 and 7 are considered as excitatory states.
body inside the water, (b) semialert with the first or two first anterior appendages contracted and half the body outside the water, and (c) alert, with all the appendages contracted and the cephalothorax outside the water (Fig. 1). Three of the displays were classified as excitatory: inactive but alert, movement of all the appendages without displacement, and fast displacement. The average time (obtained from two periods of 5 min each) that crabs were in each of the seven displays was used to determine differences between parasitized and control crabs. After the experiments, all crabs were dissected to determine the presence or absence of parasites in the hemocoel. The data on oxygen consumption and behavior were statistically analyzed using a Mann-Whitney nonparametric test (M-W) (Siegel and Castellan, 1988).
3. Results
3.1. Oxygen consumption Crabs infected with cystacanths had higher metabolic rates than control crabs (X62 SE: 0.12560.019, vs. 0.07960.014 mg O 2 l / min g, for infected and non-infected crabs, respectively; U 5 11.5, p 5 0.0013) (Fig. 2). Comparisons between the two groups indicated that their body masses were statistically similar (X62 SE: 8.71262.053; 9.12762.102, for infected and non-infected crabs, respectively; U 5 55.0, p 5 0.718).
78
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
Fig. 2. Oxygen consumption of parasitized and non-parasitized crabs expressed in mg oxygen ml / min g. Vertical lines represent 2 SE.
3.2. Behavior The parasitized crabs were significantly more active than the control crabs (80.6% vs. 35.5%; U 5 0.1 p 5 0.0003) (Fig. 3). Parasitized crabs spent more time moving all the appendages without displacement (29.2% of the total time in parasitized crabs vs. 2.2% of total time for controls; U 5 6.5, p 5 0.0027) and in fast displacement (34.2% of the total time in infected crabs, vs. 4.5% in control crabs; U 5 2; p 5 0.0007) (Fig. 4). In contrast, control crabs spent more time in resting position than parasitized ones (40.9% of the total time in control crabs vs. 5.1% in parasitized crabs; U 5 2, p 5 0.0007). For
Fig. 3. Percentage of time that control and parasitized crabs remained in activity. Vertical lines represent 2 SE.
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
79
Fig. 4. Percentage of time that control and parasitized crabs were in each of the seven recognizable behavioral displays previously described. Numbers in squares indicate excitatory attitudes. Vertical lines represent 2 SE.
the remainder of the positions or displays, there were no significant differences between both groups of crabs ( p . 0.05).
4. Discussion Thompson and Kavaliers (1994) proposed that in studies of parasitism it is possible to integrate various levels of biological organization, allowing links among behavior, physiology and ecology. Our study shows a close relationship between behavior and physiology, which supports our hypothesis that alterations of host behavior are correlated with changes in metabolic rate. Parasitized crabs had higher metabolic rates and altered activity patterns. Differences in the oxygen uptake may be due to several factors, such as type of diet, temperature, acclimation, hour of the day (circadian factors), oxygen availability, and body mass (Steffensen, 1989). The first five factors were controlled for and set constant for all crabs in the experiments. Moreover, parasitized crabs were not only more active than non-infected crabs, but also spent more time in two of the three excitatory attitudes. These excitatory attitudes, specifically movement of all appendages with or without displacement, may render crabs more visually exposed in the environment, increasing their vulnerability to predators. In Lenga estuary there are significantly higher numbers of infected crabs walking in the sand out of the water in comparison to the non-infected crabs, which usually remain on the bottom of the estuary (M. George-Nascimento, pers. comm.). This agrees well with our findings. Further, our results are consistent with the well documented hypothesis that parasites alter host behavior, which in turn may increase susceptibility of intermediate
80
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
host to predation by the final host (Holmes and Bethel, 1972; Lafferty and Morris, 1996). Visual cues are among the main stimuli for predators to detect prey. If the prey exhibits a coloration that in any way is more arresting to the predator, or if it spends more time moving about, then it may be more detectable by the predator. Profilicollis antarcticus seems to be capable of causing this type of phenotypic changes in H. crenulatus, which render the crabs more susceptible to predation and in that way the parasite may complete its development and life cycle in the new host (Pulgar et al., 1995). Pulgar et al. (1995) found that parasitized H. crenulatus presented a different pattern of carapace coloration in comparison to non-parasitized crabs. In the laboratory they measured several crab behaviors in relation to water (hydrotaxis), light (phototaxis) and presence of a simulated predator. They did not find significant differences between parasitized and control crabs. However, we think their results may be flawed for two reasons: First, the acclimation times used were very short (48 h). A period of 4 weeks is a more reasonable time for acclimation, to allow the organisms to acquire a new endocrine and nervous stable state (important for physiological and behavioral analyses). Second, crabs were artificially inoculated, so they should have done a sham inoculation (they missed a control group). Although in our study we did not test for behavioral differences correlated with special environmental stimuli (e.g., light, water), it may be expected, that parasitized crabs are more sentient than non-parasitized ones. The present study represents the first experimental demonstration that altered behaviors induced by acanthocephalan parasites in their hosts have a physiological background. Indeed, it is a physiological manipulation of the parasite by altering the metabolic rates of its host, which is expressed in behavioral changes that may increase its vulnerability to predators, which are the final hosts for the parasite.
Acknowledgements ´ We thank G. Fernandez for his constant help in keeping the oxygen consumption ´ system in good shape, and for always being there when we had problems, C.W. Caceres for his help on data analysis, and M. George-Nascimento, J. Pulgar and the rest of the members of George-Nascimento’s laboratory for the collection of crabs, and for their ˜ and S. Navarrete important comments on this work. F. Bozinovic, F. Jaksic, A. Munoz and two anonymous reviewers made helpful comments that greatly improved this manuscript. This study was funded by FONDECYT Grant 1930420.
References Beltz, B.S., Kravitz, E.A., 1986. Aminergic and peptidergic neuromodulation in Crustacea. Journal of Experimental Biology 124, 115–141. Brett, J.R., Groves, T.D., 1979. Physiological energetics. In: Hoar, W.S. (Ed.), Fish Physiology, Vol. VIII: Bioenegetics and Growth, Academic Press, New York, pp. 279–352.
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
81
Camp, J.W., Huizinga, H.W., 1979. Altered color, behavior and predation susceptibility of the isopod Asellus intermedius infected with Acanthocephalus dirus. Journal of Parasitology 65, 667–669. Carmichael, L.M., Moore, J., 1991. A comparison of behavioral alterations in the brown cockroach, Periplaneta americana, infected with the acanthocephalan Moniliformis moniliformis. Journal of Parasitology 77, 931–936. Carmichael, L.M., Moore, J., Bjostad, L.B., 1993. Parasitism and decreased response to sex pheromones in male Periplaneta americana (Dictyoptera: Blattidae). Journal of Insect Behavior 6, 25–32. Combes, C., 1991. Ethological aspects of parasite transmission. American Naturalist 138, 866–880. Dickinson, P.S., Marder, E., 1989. Peptidergic modulation of a multioscillator system in the lobster. I. Activation of the cardiac sac motor pattern by the neuropeptides proctolin and red pigment-concentrating hormone. Journal of Neurophysiology 61, 833–844. Dobson, A.P., 1988. The population biology of parasite-induced changes in host behavior. The Quaterly Review of Biology 63, 139–165. Flamm, R.E., Harris-Warrick, R.M., 1986. Aminergic modulation in lobster stomatogastric ganglion. II. Target neurons of dopamine, octopamine, and serotonine within the pyloric circuit. The American Physiological Society 55, 866–881. Gotelli, N.J., Moore, J., 1992. Altered behavior in a cockroach–acanthocephalan association. Animal Behavior 43, 949–959. Hechtel, L.J., Johnson, C.L., Juliano, S.A., 1993. Modification of antipredator behavior of Caecidotea intermedius by its parasite Acanthocephalus dirus. Ecology 74, 710–713. Holmes, J.C., Bethel, W.B., 1972. Modification of intermediate host behaviour by parasites. Zoological Journal of the Linnean Society 51, 123–149. Holmes, J.C., Zohar, S., 1990. Pathology and host behaviour by parasites. In: Barnard, C.J., Behnke J.M. (Eds.), Parasitism and Host Behaviour, Taylor and Francis, New York, pp. 34–63. Kravitz, E.A., 1988. Hormonal control of behaviour: Amines and the biasing of behavioural output in lobster. Science 241, 1775–1781. Lafferty, K.D., Morris, A.K., 1996. Altered behavior of parasitized killfish increases susceptibility to predation by bird final hosts. Ecology 77, 1390–1397. Livingstone, M.S., Harris-Warrick, R.M., Kravitz, E.A., 1980. Serotonine and Octopamine produce opposite postures in lobsters. Science 208, 76–79. Lo Bue, C.P., Bell, M.A., 1993. Phenotypic manipulation by the cestode parasite Schistocephalus solidus of its intermediate host Gasterosteus aculeatus, the threespine stickleback. American Naturalist 142, 725–735. Lozano, G.A., 1991. Optimal foraging theory: A possible role for parasites. Oikos 60, 391–395. Moore, J., 1983. Responses of an avian predator and its isopod prey to an acanthocephalan parasite. Ecology 64, 1000–1015. Moore, J., 1984a. Altered behavioral responses in intermediate host—an acanthocephalan parasite strategy. American Naturalist 123, 572–577. Moore, J., 1984b. Parasites that change the behavior of their hosts. Scientific American 250, 108–115. Moore, J., 1987. Some roles of parasitic helminths in trophic interactions. A view from North America. Revista Chilena de Historia Natural 60, 159–179. Moore, J., 1995. The behavior of parasitized animals. BioScience 45, 89–96. Moore, J., Gotelli, N.J., 1990. A phylogenetic perspective on the evolution of altered host behaviours: A critical look at the manipulation hypothesis. In: Barnard, C.J., Behnke J.M. (Eds.), Parasitism and Host Behaviour, Taylor and Francis, New York, pp. 193–229. Moore, J., Gotelli, N.J., 1992. Moniliformis moniliformis increases cryptic behaviors in the cockroach Supella longipalpa. Journal of Parasitology 78, 49–53. Moore, J., Gotelli, N.J., 1996. Evolutionary patterns of altered behavior and susceptibility in parasitized hosts. Evolution 50, 807–819. Moore, J., Lasswell, J., 1986. Altered behavior in isopods (Armadillidium vulgare) infected with the nematode Dispharynx nasuta. Journal of Parasitology 72, 186–189. Oetinger, D.F., Nickol, B.B., 1981. Effects of acanthocephalans on pigmentation of freshwater isopods. Journal of Parasitology 67, 672–684. Oetinger, D.F., Nickol, B.B., 1982. Developmental relationships between acanthocephalans and altered pigmentation in freshwater isopods. Journal of Parasitology 68, 463–469.
82
P. A. Haye, F. .P. Ojeda / J. Exp. Mar. Biol. Ecol. 228 (1998) 73 – 82
Poulin, R., 1994. Parasite manipulation of host behaviour: Should hosts always lose?. Oikos 70, 479–484. Pulgar, J., Aldana, M., Vergara, E., George-Nascimento, M., 1995. La conducta de la jaiba estuarina ´ al parasitismo por el acantocefalo ´ Hemigrapsus crenulatus (Milne-Edwards 1837) en relacion Profilicollis antarcticus (Zdzitowiecki 1985) en el sur de Chile. Revista Chilena de Historia Natural 68, 439–450. Rajashekhar, K.P., Wilkens, J.L., 1991. Dopamine and nicotine, but not serotonine, modulate the crustacean ventilatory pattern generator. Journal of Neurobiology 29, 680–691. Siegel, S., Castellan, N.J., 1988. Nonparametric statistics for the behavioral sciences, 2nd ed., McGraw-Hill, New York. Steffensen, J.F., 1989. Some errors in respirometry of aquatic breathers: How to avoid and correct for them. Fish Physiology and Biochemistry 6, 49–50. Stamp, N., 1981. Behavior of parasitized aposematic caterpillars: Advantageous to the parasitoid or the host?. American Naturalist 118, 715–725. Thompson, S.N., Kavaliers, M., 1994. Physiological bases for parasite-induced alterations of host behaviour. Parasitology 109, 119–138. Webber, R.A., Rau, M.E., Lewis, D.J., 1987a. The effects of Plagiorchis noblei (Trematoda: Plagiorchiidae) metacercariae on the behavior of Aedes aegypti larvae. Canadian Journal of Zoology 65, 1340–1342. Webber, R.A., Rau, M.E., Lewis, D.J., 1987b. The effects of Plagiorchis noblei (Trematoda: Plagiorchiidae) metacercariae on the susceptibility of Aedes aegypti larvae to predation by guppies (Poecilia reticulata) and meadow voles (Microtus pennsylvanicus). Canadian Journal of Zoology 65, 2346–2348. Zachary, A., Moore, J., Gotelli, N.J., 1992. Moniliformis moniliformis infection has no effect on some behaviors of the cockroach Diploptera punctata. Journal of Parasitology 78, 524–526.