Directory UMM :Data Elmu:jurnal:J-a:Journal of Experimental Marine Biology and Ecology:Vol245.Issue2.Mar2000:

L

Journal of Experimental Marine Biology and Ecology 245 (2000) 277–292

www.elsevier.nl / locate / jembe

Alteration of photoresponses involved in diel vertical

migration of a crab larva by fish mucus and degradation

products of mucopolysaccharides

*

Richard B. Forward Jr. , Dan Rittschof

Duke University Marine Laboratory, 135 Duke Marine Lab Rd., Beaufort, NC 285166, USA Received 20 July 1999; received in revised form 11 November 1999; accepted 18 November 1999

Abstract

Photoresponses involved in the descent phase of nocturnal diel vertical migration (DVM) of larvae of the crab Rhithropanopeus harrisii were measured in a laboratory system that mimicked the underwater angular light distribution. The test hypothesis was that kairomones from fish that activate zooplankton photoresponses involved in DVM are derived from polysaccharides from the external mucus of fishes. Studies considered fish mucus from the mummichog (Fundulus

heteroclitus) and disaccharides (originating from chondroitin sulfate A and heparin

polysac-charides) that are likely constituents of fish mucus. R. harrisii larvae descend at sunrise with an isolume and remain near the isolume during the day. Since depth maintenance near the isolume depends upon a negative phototaxis, the lowest light intensity (threshold) that induces this response was used to quantify the effects of the test chemicals. It was predicted that exposure to fish kairomones would lower the photoresponse threshold, thereby resulting in larvae remaining deeper in the water column where light for visual predation was reduced. The photoresponse threshold declined as the concentration of fish mucus increased. Disaccharides originating from chondroitin sulfate A and heparin also decreased the photoresponse threshold as compared to responses in aged, filtered seawater. Collectively, the results support the hypothesis and indicate that disaccharide degradation products of predator mucus containing sulfated and acetylated amines can serve as kairomones.  2000 Elsevier Science B.V. All rights reserved.

Keywords: Aminosugars; Chondroitin sulfate A; Crab; Diel vertical migration; Fish mucus; Glycosamino-glycans; Heparin; Kairomone; Larvae; Photoresponses; Rhithropanopeus harrisii

*Corresponding author. Tel.:11-252-504-7610; fax11-252-504-7648.

E-mail address: rforward@duke.edu (R.B. Forward Jr.)

1. Introduction

Nocturnal diel vertical migration (DVM) is commonly observed among zooplankton and consists of a single daily ascent with minimum depth reached between sunset and sunrise and a descent with maximum depth attained during the day (Forward, 1988). The most accepted hypothesis for functional significance of DVM is that zooplankton descend to dim lit areas during the day to avoid visual predators (Zaret and Suffern, 1976; Stich and Lampert, 1981) and ascend to feed during times of low light levels near the surface (reviewed by Pearre, 1979).

Field (e.g. Dini and Carpenter, 1988, 1991; Ringelberg et al., 1991) and laboratory studies (Dawidowicz et al., 1990; Tjossem, 1990; Loose, 1993) found that DVM was affected by predators. DVM was absent or reduced when predators were absent and well developed in their presence. Rapid changes in DVM in the presence and absence of predators (Bollens and Frost, 1989b, 1991; Ringelberg et al., 1991; Neill, 1992) indicate that a phenotypic response occurs in which zooplankton behavior and physiology are modified by cues from planktivors. With the exception of Bollens and Frost’s (1989a) study, evidence indicates prey use chemical cues (kairomones) to detect predators (e.g. Dodson, 1990; Neill, 1990, 1992; Ringelberg, 1991a,b; Loose, 1993; Loose et al., 1993). Since behavioral responses to light underlie DVM, photoresponses are modified by kairomones from predators, such as planktivorous fishes (e.g. Ringelberg, 1995; Ringelberg and Van Gool, 1995; Van Gool and Ringelberg, 1995; Van Gool, 1998).

Recently, Forward and Rittschof (1999) hypothesized that fish kairomones include disaccharide degradation products of polysaccharides from the external mucus of fishes. Mucus is composed of protein moieties (5% of dry weight) covalently bonded to polysaccharide chains (95% of dry weight), which are termed glycosaminoglycans (GAGs; Shephard, 1994). The chains are composed of repeating disaccharide units, each with a molecular weight of 400–600 Da (Brimacombe and Weber, 1964; Fransson, 1985). In general, the four main types of GAGs are (1) hyaluronic acid, (2) chondroitin sulfate and dermatan sulfate, (3) heparan sulfate and heparin, (4) keratan sulfate. Early studies indicated that GAGs are present in fish mucus (Wasserman et al., 1972; Wold and Selset, 1977) but the specific types of GAGs were not identified. More recently, chondroitin sulfate and hyaluronic acid have been identified in fish mucus (van de Winkel et al., 1986; Karamanos et al., 1991), and secretory products of fish epithelial mucus cells produce polysaccharides with terminal groups found in chondroitin sulfate (Hidalgo et al., 1987). The other two major classes of GAGs (keratan sulfate and heparin) are not reported in fish mucus.

Forward and Rittschof (1999) used photoresponses involved in the descent phase of nocturnal DVM of brine shrimp larvae to assess the activity of fish mucus and polysaccharide subunits. Photoresponses were activated by (1) fish mucus at

con-26 21

centrations above 10 g wet weight l and (2) disaccharide subunits of heparin, chondroitin sulfate A and hyaluronic acid. Subunits with the most potent biological activity had either a sulfamino or acetylamino group on carbon 2 of the hexosamine. The present study was designed to determine whether the results with brine shrimp larvae apply to another zooplankter.

sunrise, larvae move down with the isolume that corresponds to the lowest light intensity for phototaxis (Forward et al., 1984) and remain with this isolume during the day. The behavioral responses underlying this relationship are a negative geotaxis in darkness, which changes to negative phototaxis in light (Forward et al., 1984; Forward, 1985). Thus, larvae oscillate around an isolume by swimming up in darkness and reversing direction when they encounter perceptible light. In contrast, the migratory ascent at sunset is initiated by the relative rate of light intensity decrease (Forward, 1985).

Since zooplankton descend at sunrise and remain at depth during the day to avoid visual predators, such as fish, (e.g. Zaret and Suffern, 1976; Stich and Lampert, 1981), the underlying photoresponses of R. harrisii larvae were used to assess the activity of fish kairomones. The lowest light intensity to induce the negative phototaxis (photo-response threshold) will determine the isolume with which larvae migrate vertically. It was predicted that the threshold would be lower in the presence of fish kairomones, thereby moving larvae deeper in the water column during the day where there would be less light for visual predation. Alternatively, if kairomones are not present, the depth of migration will be reduced because the photoresponse threshold will be higher. In this way, the daytime depth of larvae would be graded and increase as the concentration of fish kairomones increases.

The test hypothesis was that kairomones from fish that alter photoresponses involved in DVM are derived from polysaccharides comprising their external mucus. The components of fish mucus that altered R. harrisii larval photoresponses were character-ized by testing (1) fish mucus, (2) chondroitin sulfate A and heparin disaccharides and (3) subunits of these disaccharides. These disaccharides were tested because (1) chondroitin sulfate occurs in fish mucus (van de Winkel et al., 1986; Hidalgo et al., 1987; Karamanos et al., 1991), (2) heparin disaccharides activated photoresponses involved in DVM of brine shrimp larvae (Forward and Rittschof, 1999) and (3) heparin disaccharide stimulate alarm behavioral responses in snails that are similar to responses to fish predator odor (Rahman et al., 1999). Testing these substances would provide the most concise information for assessing whether the results with brine shrimp larvae (Forward and Rittschof, 1999) apply to another zooplankter.

The major repeating disaccharide unit of heparin is uronic acid with a sulfate at the 2 carbon position coupled by an alpha 1–4 glycoside linkage to D-glucosamine having a

sulfate ester on carbon 6 and a sulfamino group on carbon 2 (Fig. 1). The sulfamino group is unique to heparin and heparan sulfate. The major disaccharide in chondroitin sulfate A (Fig. 1) is uronic acid coupled by an alpha 1–3 glycoside linkage to

D-galactose amine, which has a sulfated ester on carbon 4 and an acetylamine on carbon

2. The results support the hypothesis and agree with the brine shrimp study (Forward and Rittschof, 1999) because both fish mucus and these disaccharides alter photo-responses of R. harrisii larvae.

2. Materials and methods

2.1. Larval rearing and photoresponse techniques

Fig. 1. Heparin and chondroitin sulfate A disaccharides with positions (R) for sulfated esters and hydrogen groups. The carbons on each ring are numbered 1 to 6. The different R groups are also numbered (e.g. R1) for reference in the text.

6-h interval in the middle of the light phase to avoid complications due to possible biological rhythms in behavior.

On the day of testing, each brood of larvae was divided into 3–4 equal groups depending on numbers. One group was tested for photoresponses when exposed to clean seawater and the other groups were exposed to different chemical treatments added to clean seawater. Larvae were only tested after exposure to one chemical treatment and never used again. All experiments were replicated five times using larvae from different females.

Larvae were tested in a room maintained at about 248C in an apparatus designed to produce a light field similar to that occurring underwater during the day (see Forward et al. (1984) for a detailed description). Larvae were placed in a Lucite test chamber (53535 cm), which was positioned at the horizontal center of a much larger water bath (50350325 cm) with the inside walls painted flat black. The difference in stimulus light intensity throughout the bath was about 10%. The bath contained deionized water. The test chamber was elevated so that its walls were slightly above that of the water bath, thus preventing deionized water from entering the test chamber. The size of the bath was such that its walls were outside the critical angle (zenith648.68) as viewed from the bottom of the test chamber.

The light stimulus system was a 300 W incandescent lamp filtered to 500 nm with an interference filter (Ditric Optics; half-band pass 7.4 nm). This wavelength is the major spectral sensitivity maximum of the larvae (Forward and Cronin, 1979). Light intensity was controlled by fixed neutral density filters (Oriel Corp.) and intensity measured with a radiometer (EG&G; model 550). During experimentation larvae were illuminated with far-red light (maximum transmission 775 nm) and observed with a video system (Forward, 1985). Photoresponses are not altered or induced by far-red light (Forward and Cronin, 1979).

Each experiment began by placing Stage IV zoea in the test solutions (see below) for a minimum of 3 h. Tests with other crustacean larvae indicated that activation of photoresponses involved in DVM by fish kairomones occurs within 1 h (Forward and Rittschof, 1993; McKelvey and Forward, 1995). After treatment, larvae were transferred

21

to the test chamber in the water bath (density 1–4 ml ) and adapted to darkness for at least 1 h. After dark adaptation, larvae were stimulated with light for 3 s at 4-min

11

intervals. Each group of larvae received 10 stimuli ranging in intensity from 2.9310

11 22 21

to 46.6310 photons m s that were presented in ascending order. Larvae returned to their pre-stimulation swimming pattern and photoresponse level within the 4-min interval between stimuli (Forward et al., 1984). Thus, photoresponsiveness remained consistent with repetitive stimulation at different stimulus levels. Preliminary

experi-11 22 21

ments found that 2.9310 photons m s was the lowest light intensity to evoke a descent photoresponse (see below) when larvae were exposed to test chemicals.

The percentage of larvae descending was calculated for each trial at each stimulus condition. Means, standard deviations and standard errors for combined trials were calculated after data were arcsine transformed. Back-transformed means and standard errors are plotted on the figures. For each test chemical, relative changes in the percentage of larvae descending before and after light stimulation were compared using a Friedman’s test (Zar, 1984). A one-tailed statistical test was conducted, since light stimulation was expected to increase the percent descending. If the Friedman’s test indicated a significant difference among light stimulation levels, the lowest light intensity to evoke a significant descent response (threshold) was determined by comparing relative changes in percent descending to control values using a Wilcoxon paired-sample test (one tailed) at an experimental-wise error rate of 0.05.

2.2. Test chemicals

Experiments were designed to test larval photoresponses following exposure to specific concentrations of specific test chemicals. Greater activation of photoresponses involved in DVM was indicated by a reduction in the lowest light intensity (threshold) that evoked a significant descent response. The three groups of test chemicals were (1) fish mucus, (2) commercially purified heparin and chondroitin sulfate A disaccharides (Sigma), and (3) commercially purified disaccharide subunits (Sigma). Fish mucus and chemical concentrations were based on the previous study with brine shrimp larvae (Forward and Rittschof, 1999).

2.2.1. Clean seawater

Clean seawater was prepared by septic filtration of water from the Newport River estuary to remove biologically active molecules larger than 100 kDa, followed by aging for a minimum of 1 week. This procedure removed biologically active molecules and produced water with a consistent chemical composition (e.g. Rittschof et al., 1983). Clean seawater did not activate photoresponses involved in DVM of brine shrimp larvae (Forward and Hettler, 1992; Forward and Rittschof, 1993; McKelvey and Forward, 1995; Forward and Rittschof, 1999). All test solutions were made up in clean seawater

26

on the day of testing. Specific chemicals were tested at a single concentration of 10 M because a previous study (Forward and Rittschof, 1999) found that chemicals, which affected photoresponses were maximally active at this concentration.

2.2.2. Fish mucus



preweighed Kimwipe . Fish were exposed to air for ,30 s during the procedure and returned immediately to water. They showed no sign of disease and were healthy in appearance for the next week. The five wipes carrying the mucus from five fish were weighed again, and the amount of material removed from the surface of the fish was determined by the difference between initial and final weights. The wipes were then placed in a 100-ml beaker, to which 50 ml of clean seawater were added. After covering, the beaker was incubated at 238C on an orbital shaker at 60 rev. / min for 40 min. At the end of the incubation interval, 42.5 ml of liquid was recovered by decanting liquid from



the beaker and squeezing the Kimwipes with a latex surgical gloved hand. This

21

solution was frozen until use and had a sulfated sugar concentration of 260mg ml as determined using heparin disaccharide (Sigma [ _{H-9267) as a standard in the phenol} sulfuric procedure (Ashwell, 1955). For tests with fish mucus, larvae were incubated in clean seawater having a defined mucus concentration (g wet weight of fish mucus

21 21 21

l 5g l ) of either 0.1 or 0.001 g l . A previous study found that the activity of fish 

mucus was not due to Kimwipes (Forward and Rittschof, 1999).

2.2.3. Components structurally related to chondroitin sulfate A subunits

The repeating disaccharide in chondroitin sulfate A polysaccharide is sulfated on

2

carbon 4 of galactose (a-DUA[1→3]-GalNAc-4S; R1 and R35H, R25SO ; Fig. 1;3

Sigma [ _{C-4045), where DUA is 4-deoxy-}L-threo-hex-4-enopyranosyluronic acid, Gal

is D-galactosamine, Ac is acetyl and 4S is 4-sulfate. Experiments were done with this

disaccharide and its associated monosaccharide acetyl galactosamine, which lacks sulfates (N-acetyl-D-galactosamine; R1 and R25H; Fig. 1; Sigma [ A-2795).

2.2.4. Components structurally related to heparin subunits

Disaccharide components of heparin were tested to determine the functional groups that activate larval photoresponses. The first experiment tested the heparin disaccharide

2

(R1, R2, R35SO ; Sigma [ _{H-9267; Fig. 1). Heparin disaccharide is a-D}

UA-2S-3

[1→4]-GlcNS-6S whereDUA is 4-deoxy-L-threo-hex-4-enopyronosyluronic acid, GlcN

is D-glucosamine, NS is N sulfo, 2S is 2-sulfate and 6S is 6-sulfate. This disaccharide

would be produced by the action of heparinase lyase on the heparin polysaccharide. It differs from chondroitin sulfate A (above) by having a 1–4 disaccharide linkage and by the substitution of N-sulfamino-D-glucosamine for N-acetyl-D-galactosamine. The most

unique structural part of this molecule is the sulfamino group on the second carbon (signature sulfamino group: R2; Fig. 1) of glucosamine. Thus, the second experiment tested the heparin disaccharide that had only this sulfamino group (a-DUA-[1→

4]-2

GlcNS; R1 and R35H, R25SO ; Sigma [ _{H-1145). In order to determine whether}

3

activity of the molecule was due to the signature sulfate, the final experiment tested heparin disaccharide without any sulfates (a-DUA-[1→4]-GlcN; R1, R2 and R35H: Fig. 1; Sigma [ _H-9276).

3. Results

intensity increased. The effect of exposure to different chemicals was quantified by determining the minimum light intensity to evoke a significant descent response (threshold). Upon exposure to clean seawater, the descent response had a threshold of

11 22 21

18.3310 photons m s (Fig. 2). This threshold represented photoresponsiveness in the absence of kairomones. A decline in the threshold upon exposure to the test chemical would indicate alteration of photoresponses involved in DVM.

When exposed to fish mucus, the photoresponse threshold decreased as the fish mucus

21

concentration increased (Fig. 3). At 0.001 g wet weight l , the threshold was

11 22 21 21

11.6310 photons m s (Fig. 3B), while at 0.1 g wet weight l the threshold was

11 22 21

about an order of magnitude lower at 2.9310 photons m s (Fig. 3A). Thus, photoresponsiveness increased upon exposure to fish mucus, and the magnitude of the effect depended upon concentration.

Considering components of fish mucus, the chondroitin sulfate A polysaccharide is composed of repeating disaccharide groups, in which uronic acid is connected to galactosamine by a 1–3 glycoside linkage. The repeating disaccharide has a sulfate ester on carbon 4 of galactosamine and an N-acetyl group on carbon 2 (Fig. 1; a-D

UA-26

[1→3]-GalNAc-4S). At a concentration of 10 M, this disaccharide induced photo-responses that were similar to those upon exposure to the highest concentration of fish

Fig. 3. Percentage of larvae showing a descent response upon stimulation with different light intensities when

21 21

exposed to fish mucus at concentrations of 0.1 g wet weight l (A) and 0.001 g wet weight l (B). The solid line (solid circles) shows responses upon light stimulation, while the dashed line (open circles) shows the percent descending prior to stimulation (control). The asterisk indicates the lowest light intensity that evokes a descent response significantly (P,0.05; Wilcoxon pair-sample test) greater than the control level (threshold). The sample size was five. Means and standard errors are plotted.

11 22 21

mucus (Fig. 3A), since the threshold was 2.9310 photons m s (Fig. 4A). The active part of the molecule is probably the acetyl galactosamine because the monosac-charide N-acetylD-galactosamine at a similar concentration induced photoresponses with

Fig. 4. Percentage of larvae showing a descent response upon stimulation with different light intensities when exposed to (A) chondroitin sulfate A disaccharide (a-DUA-[1→3]-GalNAc-4S) and (B) N-acetyl D

-galac-26

tosamine at a concentration of 10 M. The solid line shows responses upon light stimulation, while the dashed line shows the percent descending prior to stimulation (control). The asterisk indicates the lowest light intensity that evokes a descent response significantly (P,0.05; Wilcoxon pair-sample test) greater than the control level (threshold). The sample size was five. Means and standard errors are plotted.

Heparin polysaccharides are composed of repeating disaccharide groups of uronic acid connected by an alpha 1–4 glycoside linkage to N-sulfoD-glucosamine. Uronic acid has

sulfated esters on carbon 2 while D-glucosamine has a sulfate ester on carbon 6 and a 2

4]-GlcNS-26

6S). At 10 M (Fig. 5A), this disaccharide caused an increase in responsiveness as the

11 22 21

threshold (5.8310 photons m s ) was lower than that in clean seawater. The distinctive part of this disaccharide is the sulfamino moiety at the 2 carbon position

2

(R25SO ; Fig. 1). The heparin disaccharide with only this signature sulfamino group3

(a-DUA-[1→4]-GlcNS) was more potent than the parent molecule, as the threshold

11 22 21

declined to 2.9310 photons m s (Fig. 5B). If all of the sulfates were removed (R15H; R25H; R35H; Fig. 1;a-DUA-[1→4]-GlcN) the molecule had low activity,

11 22

since the threshold was very close to that for clean seawater at 14.5310 photons m

21

s (Fig. 5C). Collectively, these results indicate that most of the activity of the heparin disaccharide can be attributed to the signature sulfamino group.

4. Discussion

Field studies indicate that larvae of the crab Rhithropanopeus harrisii undergo nocturnal diel vertical migration (DVM), in which they descend at sunrise and ascend at sunset (Cronin, 1982; Forward et al., 1984). Recent studies found that invertebrate zooplankton have a phenotypic response, in which DVM is activated by kairomones from fish predators and absent or reduced in the absence of these kairomones (e.g. Dodson, 1990; Neill, 1990, 1992; Ringelberg, 1991a,b; Loose, 1993; Loose et al., 1993). Thus, the test hypothesis for the present study was that photoresponses of R. harrisii larvae that are involved in DVM are altered by kairomones from fish, which consist of disaccharide degradation products of polysaccharides that make up their external mucus.

R. harrisii larvae descend with an isolume at sunrise and remain near the isolume during the day (Forward et al., 1984). If a function of the descent is to avoid visual predators (Zaret and Suffern, 1976; Stich and Lampert, 1981), then larvae should descend to lower light levels if predators are present and be shallower when they are absent. Since the day depth of the R. harrisii larvae is related to an isolume (Forward et al., 1984), it is predicted that they should be associated with lower intensity isolumes in the presence of kairomones from fish. The behavioral responses underlying depth maintenance at an isolume are a negative geotaxis in apparent darkness that changes to a negative phototaxis in the presence of light (Forward et al., 1984; Forward, 1985). The light intensity that evokes the negative phototaxis serves as a barrier for upward movement and would be the isolume with which larvae are associated. Thus, it was also predicted that the lowest light intensity to evoke the negative phototaxis (threshold) would be high in the absence of fish kairomones and lower in their presence.

The results support the hypothesis, as the threshold for negative phototaxis was higher

11 22 21

in the presence of clean seawater (18.3310 photons m s ; Fig. 2; Table 1) and

11 22 21

decreased by 84% (2.9310 photons m s ; Fig. 3A; Table 1) in the presence of

21

fish mucus at a concentration of 0.1 g wet weight l . Responsiveness is related to fish

21

mucus concentration. At 0.001 g wet weight l , the threshold was only reduced by 36%

11 22 21

Fig. 5. Percentage of larvae showing a descent response upon stimulation with different light intensities when exposed to (A) heparin disaccharide (a-DUA-2S-[1→4]-GlcNS-6S), (B) heparin disaccharide that lacked all sulfated esters but has the sulfamino group on the second carbon of glucosamine (a-DUA-[1→4]-GlcNS) and

Table 1

Photoresponse thresholds ordered from the highest to lowest

a

Treatment Threshold light intensity % Decrease Figure

11 22 21 (310 photons m s )

Clean seawater 18.3 2

Heparin no sulfamino 14.5 21 5C

group

21

Fish mucus (0.001 g l ) 11.6 36 3B

Heparin 5.8 68 5A

N-acetylD-galactosamine 2.9 84 4B

Chondroitin sulfate A 2.9 84 4A

Heparin with only 2.9 84 5B

sulfamino group

21

Fish mucus (0.1 g l ) 2.9 84 3A

a

% Decrease is the percent reduction in the threshold as compared to the value in clean seawater.

The test hypothesis was that kairomones originate from fish mucus. This hypothesis was supported, as exposure to fish mucus lowered the photoresponse threshold. The lowest fish mucus concentration to alter photoresponsiveness was not determined but is

21

less than 0.001 g wet weight l . For brine shrimp larvae the lowest effective

25 21

concentration was 10 g wet weight l , which had a sulfated sugar level of about 0.1

21

mg l (Forward and Rittschof, 1999). Assuming R. harrisii larvae have the same sensitivity, the lowest effective concentration can be related to mucus released from a fish that occurs with R. harrisii larvae. Parrish and Kroen (1988) quantified the rate at which mucus was sloughed from Atlantic silversides (Menidia menidia), a planktivorous fish found in marine and estuarine areas. Mucus was measured as sulfated sugar using the same technique as in the present study. Each fish released 567 mg of sulfated sugar

21

per hour when swimming at a speed of 50 cm s . Based on their measurements, a

21

school of 66 Atlantic silversides swimming at a speed of 50 cm s would leave a trail

21 24

of mucus having a sulfated sugar concentration of 0.1 mg l (10 ppm). Schools of Atlantic silversides are frequently much larger than 66 fish (D. Rittschof, personal observation).

Fish mucus is mainly composed of polysaccharide chains of repeating disaccharide units (Brimacombe and Weber, 1964; Fransson, 1985; Shephard, 1994). The di-saccharides studied originated from heparin and chondroitin sulfate A. Didi-saccharides of these polysaccharides differ in their linkage (1–3 or 1–4), and location and presence of sulfamino, acetylamino, sulfated esters and acetyl groups (Fig. 1). The past (Forward and Rittschof, 1999; Rahman et al., 1999) and present studies clearly indicate that a modified amine (sulfamino or acetylamino) is important for activity.

26

At 10 M, the disaccharide of chondroitin sulfate A induced photoresponses (Fig.

21

4A; Table 1) that were similar to those after exposure to 0.1 g fish mucus l . The major monosaccharide of chondroitin sulfate A (N-acetyl D-galactosamine) was equally active

kairomone. Hyaluronic acid is a structurally similar polysaccharide and also occurs in fish mucus (van de Winkel et al., 1986; Karamanos et al., 1991). Activity of its disaccharide remains to be tested with crab larvae, but it did activate photoresponses involved in DVM of brine shrimp larvae (Forward and Rittschof, 1999).

Heparin disaccharide was moderately active, as the photoresponse threshold declined

11 22 21

68% to 5.82310 photons m s as compared to that in clean seawater (Fig. 5A; Table 1). If all sulfated esters were removed from the disaccharide except the signature sulfamino group at the 2 carbon position (Fig. 1), activity increased as the threshold

11 22 21

declined by 84% to 2.9310 photons m s (Fig. 5B; Table 1). This sulfamino group is important for activity because the disaccharide lacking this group induced only

11 22 21

a 20% decline in the photoresponse threshold (14.5310 photons m s ; Fig. 5C; Table 1). Heparin is not reported in fish mucus. If it is not a component of fish mucus, its activity may result from its structural similarity to chondroitin sulfate A.

Collectively, these results support the hypothesis that degradation products of polysaccharides, which make up the external mucus of fishes serve as kairomones that alter photoresponses of R. harrisii larvae involved in DVM. Although studies of brine shrimp larvae (Forward and Rittschof, 1999) and of snail alarm behavior (Rahman et al., 1999) also supported this hypothesis, a number of future studies are needed to verify that degradation products from fish mucus serve as fish kairomones and to identify other types of compounds that also serve as kairomones. Specific polysaccharides and their breakdown products should be identified in water containing fish. The levels of these products in natural waters need to be measured and related to DVM. The activity of other kairomones could be indicated by removing saccharides from water in which fish were incubated and test the remaining water for effects on photoresponses. To determine if degradation products of mucopolysaccharides also serve as kairomones for other zooplankton, fish mucus degradation products need to be tested on other zooplankton species from different environments (e.g. marine and freshwater).

Degradation products of fish mucus are well suited as fish kairomones because mucus is sloughed continuously as they swim. Thus, mucus degradation products are produced continuously. The unique and unstable nature of modified amines of the disaccharides make them ideal candidates as specific signal molecules because they have a low signal to noise ratio, a short half life, and highly predictable occurrence. These properties are comparable to those of peptide signal molecules like the arginine carboxyl terminal peptides that have a wide range of physiological, signaling and pheromone functions (e.g. Rittschof, 1985, 1993; Zimmer-Faurst and Tamburri, 1994). These features and the fact that representatives of at least two phyla of zooplankton respond to fish mucus breakdown products and pure disaccharides suggest that modified amino sugars may have a broad variety of signal functions.

Acknowledgements

References

Ashwell, G., 1955. The phenol–sulphuric acid reaction for carbohydrate. Methods Enzymol. 8, 93–94. Bollens, S.M., Frost, B.W., 1989a. Zooplankton fish and variable diel vertical migration in the marine

planktonic copepod Calanus pacificus. Limnol. Oceanogr. 34, 1072–1083.

Bollens, S.M., Frost, B.W., 1989b. Predator-induced diel vertical migration in a planktonic copepod. J. Plankton Res. 11, 1047–1065.

Bollens, S.M., Frost, B.W., 1991. Diel vertical migration in zooplankton: rapid individual response to predators. J. Plankton Res. 13, 1359–1365.

Brimacombe, J.S., Weber, J.M., 1964. Mucopolysaccharides, Elsevier, Amsterdam.

Cronin, T.W., 1982. Estuarine retention of larvae of the crab Rhithropanopeus harrisii. Estuar. Coast. Shelf Sci. 15, 207–220.

Dawidowicz, P., Pijanowska, J., Ciechomski, K., 1990. Vertical migration of Chaoborus larvae is induced by the presence of fish. Limnol. Oceanogr. 35, 1631–1637.

Dini, M.L., Carpenter, S.R., 1988. Variability in Daphnia behavior following fish community manipulation. J. Plankton Res. 10, 621–635.

Dini, M.L., Carpenter, S.R., 1991. The effect of whole-lake fish community manipulations on Daphnia migratory behavior. Limnol. Oceanogr. 36, 370–377.

Dodson, S., 1990. Predicting diel vertical migration of zooplankton. Limnol. Oceanogr. 35, 1195–1200. Forward, Jr. R.B., 1985. Behavioral responses of larvae of the crab Rhithropanopeus harrisii (Brachyura:

Xanthidae) during diel vertical migration. Mar. Biol. 90, 9–18.

Forward, Jr. R.B., 1988. Diel vertical migration: zooplankton photobiology and behavior. Oceanogr. Mar. Biol. Annu. Rev. 26, 361–392.

Forward, R.B., Cronin, T.W., 1979. Spectral sensitivity of larvae from intertidal crustaceans. J. Comp. Physiol. 133, 311–315.

Forward, Jr. R.B., Hettler, Jr. W.F., 1992. Effects of feeding and predator exposure on photoresponses during diel vertical migration of brine shrimp larvae. Limnol. Oceanogr. 37, 1261–1270.

Forward, Jr. R.B., Rittschof, D., 1993. Activation of photoresponses of brine shrimp nauplii involved in diel vertical migration by chemical cues from fish. J. Plankton Res. 15, 693–701.

Forward, R.B., Jr., Rittschof, D., 1999. Brine shrimp larval photoresponses involved in diel vertical migration: activation by fish mucus and modified amino sugars. Limnol. Oceanogr., in press.

Forward, Jr. R.B., Cronin, T.W., Stearns, D.E., 1984. Control of diel vertical migration: photoresponses of a larval crustacean. Limnol. Oceanogr. 29, 146–154.

Fransson, L., 1985. Mammalian glycosaminoglycans. In: Aspinall, G.O. (Ed.), The Polysaccharides, Academ-ic, New York, pp. 338–417.

Frost, B.W., 1988. Variability and possible adaptive significance of diel vertical migration in Calanus pacificus, a planktonic marine copepod. Bull. Mar. Sci. 43, 675–694.

Hidalgo, J., Velasco, A., Sanchez-Aguayo, I., Amores, P., 1987. Light microscopic characterization of glycoconjugates in secretory cells of the carp (Cyprinus carpio) gill epithelium. Histochemistry 88, 65–74. Karamanos, N.K., Manouras, A., Politou, D., Gritsoni, P., Tsegendidis, T., 1991. Isolation and high-performance liquid chromatographic analysis of ray (Raja clavata) skin glycosaminoglycans. Comp. Biochem. Physiol. 100B, 827–832.

Loose, C.J., 1993. Daphnia diel vertical migration behavior: response to vertebrate predator abundance. Arch. Hydrobiol. Beih. Ergebn. Limnol. 39, 29–36.

Loose, C.J., Von Elert, E., Dawidowicz, P., 1993. Chemically-induced diel vertical migration in Daphnia: a new bioassay for kairomones exuded by fish. Arch. Hydrobiol. 126, 329–337.

McKelvey, L.M., Forward, Jr. R.B., 1995. Activation of brine shrimp nauplii photoresponses involved in diel vertical migration by chemical cues from visual and non-visual predators. J. Plankton Res. 17, 2191–2206. Neill, W.E., 1990. Induced vertical migration in copepods as a defense against invertebrate predation. Nature

345, 524–525.

Neill, W., 1992. Population variation in the ontogeny of predator-induced vertical migration of copepods. Nature 356, 54–57.

Parrish, J.K., Kroen, W.K., 1988. Sloughed mucus and drag-reduction in a school of Atlantic silversides,

Pearre, Jr. S., 1979. Problems of detection and interpretation of vertical migration. J. Plankton Res. 1, 29–44. Rahman, Y.J., Forward, R.B., Jr., Rittschof, D., 1999. Responses of mud snails and perwinkles to

environmental odors and disaccharide mimics of fish odor. Mar. Freshwater Behav. Physiol., in press. Ringelberg, J., 1991a. A mechanism of predator-mediated induction of diel vertical migration in Daphnia

hyalina. J. Plankton Res. 13, 83–89.

Ringelberg, J., 1991b. Enhancement of the phototactic reaction in Daphnia hyalina by a chemical mediated by juvenile perch (Perca fluviatilis). J. Plankton Res. 13, 17–25.

Ringelberg, J., 1995. Changes in light intensity and diel vertical migration: a comparison of marine and freshwater environments. J. Mar. Biol. Assoc. UK 75, 15–25.

Ringelberg, J., Van Gool, E., 1995. Migrating Daphnia have a memory for fish kairomones. Mar. Fresh. Behav. Physiol. 26, 249–257.

Ringelberg, J., Flik, B.J., Lindenaar, D., Royackers, K., 1991. Diel vertical migration of Daphnia hyalina (sensulatiori) in Lake Maarssenveen: Part 1. Aspects of seasonal and daily timing. Arch. Hydrobiol. 121, 129–145.

Rittschof, D., 1985. Oyster drills and the frontiers of chemical ecology: unsettling ideas. Am. Malacol. Bull. Spec. Ed. 1, 111–116.

Rittschof, D., 1993. Body odors and neutral-basic peptide mimics: a review of responses by marine organisms. Am. Zool. 33, 487–493.

Rittschof, D., Williams, L.G., Brown, B., Carriker, M.R., 1983. Chemical attraction of newly hatched oyster drills. Biol. Bull. 164, 493–505.

Shephard, K.L., 1994. Functions for fish mucus. Rev. Fish Biol. Fish. 4, 401–429.

Stich, H.-B., Lampert, W., 1981. Predator evasion as an explanation of diurnal vertical migration by zooplankton. Nature 293, 396–398.

Tjossem, S.F., 1990. Effect of fish chemical cues on vertical migration behavior of Chaoborus. Limnol. Oceanogr. 35, 1456–1468.

van de Winkel, J.G.J., Toin, H.M.S., van Kupevelt, M., Janssen, H.M.J., Lock, R.A., 1986. Glycosamino-glycans in the skin of rainbow trout (Salmo gairdneri ). Comp. Biochem. Physiol. 85B, 473–475. Van Gool, E., 1998. In: Diel Vertical Migration of Daphnia: an Inquiry into Mechanisms for Phototactic

Behaviour. Ph.D. Dissertation, University of Amsterdam, Netherlands, pp. 1–117.

Van Gool, E., Ringelberg, J., 1995. Swimming of Daphnia galeata3hyalina in response to changing light

intensity: influence of food availability and predator kairomone. Mar. Fresh. Behav. Physiol. 26, 259–265. Wasserman, L., Ber, A., Allalouf, D., 1972. Acidic glycosaminoglycan composition of the gills of Cyprinus

carpio. Comp. Biochem. Physiol. 42B, 669–677.

Wold, J.K., Selset, R., 1977. Glycoproteins in the skin mucus of the char (Salmo alpinus L.). Comp. Biochem. Physiol. 56B, 215–218.

Zar, J.H., 1984. Biostatistics, Prentice-Hall, Englewood Cliffs, NJ.

Zaret, R.M., Suffern, S.S., 1976. Vertical migration in zooplankton as a predator avoidance mechanism. Limnol. Oceangr. 21, 804–813.