Serotonin and dopamine have opposite effects on phototaxis in larvae of the bryozoan Bugula neritina.
Regulation of the vertical distributions of pelagic larvae of marine invertebrates is most likely restricted to the vectoral factors of current velocity, light direction, and gravity (Crisp, 1984). Of these, most is known about the influence of light. Studies of the role of phototaxis in larval behavior were pioneered by Thorson (1964), who documented the photic responses exhibited by larvae of 141 species of shallow-water benthic marine invertebrates from 11 phyla. In Thorson's survey, the most frequently observed response to light was for larvae to be initially positively phototactic (82%). Only 6% responded negatively to light throughout larval life, and only 12% were indifferent to light. In 76% of the cases examined, larvae that were initially photopositive became photonegative before the end of the larval period. Apparently, an ontogenetically regulated switch governing the sign of phototaxis is found in larvae of many species. Positive phototaxis early in the larval period may bring larvae up into the water column and facilitate dispersal; later negative phototaxis may bring them down to the substratum where settlement occurs. In addition to changes in phototaxis during development, environmental signals may influence phototactic behavior of larvae (see Crisp, 1984; Young and Chia, 1987, for reviews). Factors such as temperature, salinity, light intensity, ionic shock, pH, exposure to chemical cues from settlement-specific substrates, and pollutants all have been implicated in environmentally induced changes in phototaxis.
Despite the wide phylogenetic distribution and ecological significance of larval phototaxis, very little is known about the internal mechanisms responsible for generating and modulating this behavior. This lack of knowledge stands in contrast to current understanding of the neural bases of stereotyped behaviors in many adult invertebrates. Although that literature is far too extensive to review here, it is important to point out a common theme that has emerged in the last two decades: Many behaviors are initiated, maintained, altered, or terminated by the action of monoamine or peptide modulators on neural networks (see reviews by Harris-Warrick and Marder, 1991; Katz, 1995). Examples involving monoamines include initiation of swimming in an annelid (Willard, 1981; Mangan et al., 1994); modulation of phototaxis, swimming, respiration, and feeding in gastropods (Crow and Forrester, 1986; McClellan et al., 1994; Syed et al., 1990; Wieland and Gelperin, 1983; Kyriakides and McCrohan, 1989); starting and stopping flight in an insect (Claassen and Kammer, 1985); and switching of body postures and modulation of pyloric motor output in decapod crustaceans (Livingstone et al., 1980; Flamm and Harris-Warrick, 1986).
Although evidence has been presented for the existence of neuroactive monoamines in the larvae of many marine invertebrates - including hydrozoans (McCauley, 1995; Walther et al., 1996), a nemertean (Hay-Schmidt, 1990a), a polychaete (Hay-Schmidt, 1995), bivalve and gastropod molluscs (Coon and Bonar, 1986; Goldberg and Kater, 1989; Marois and Carew, 1990; Barlow and Truman, 1992; Pires et al., 1992), several echinoderms (Toneby, 1980; Burke, 1983; Burke et al., 1986; Bisgrove and Burke, 1986, 1987; Nakajima, 1987, 1988; Thorndyke et al., 1992), brachiopods (Hay-Schmidt, 1992), phoronids (Hay-Schmidt, 1990b,c), a hemichordate (Dautov and Nezlin, 1992), and a cephalochordate (Holland and Holland, 1993) - less is known of the roles of these compounds in the mediation or modulation of larval behaviors. Monoamines have been implicated in the control of metamorphosis in a variety of taxa including hydrozoans (McCauley, 1995; Walther et al., 1996), polychaetes (Biggers and Laufer, 1992; Okamoto et al., 1995), gastropods (Couper and Leise, 1996; Pires et al., 1995), bivalves (Coon and Bonar, 1987; Bonar et al., 1990; Chevolot et al., 1991; Kingzett et al., 1990), a barnacle (Yamamoto et al., 1996), and an echinoid (Burke, 1983). Effects of monoamines on ciliary locomotion have also been reported. Bath-applied dopamine (DA) induces ciliary reversal and backward swimming in some echinoid plutei (Lacalli and Gilmour, 1990; Mogami et al., 1992), while serotonin (5HT) increases the speed of forward swimming (Mogami et al., 1992). Serotonin also accelerates ciliary beat frequency in encapsulated embryos of the gastropod Helisoma trivolvis, while DA has no effect (Diefenbach et al., 1991; Goldberg et al., 1994). However, no data have been published on the regulation of larval phototaxis by monoamines or any other neuroactive substances.
Bryozoans are excellent material for experimental studies of larval phototaxis because most species retain their early developmental stages and, on stimulus of light, release larvae that generally settle within a few hours of eclosion. Cohorts of larvae all released within a few minutes of each other can thus be obtained. Ryland (1976, 1977) reviewed in detail the early studies of the phototactic behavior of bryozoan larvae, and several important conclusions come from these studies. First, a range of responses to light are possible, depending on the species. During the course of its larval existence an individual could remain neutral to light throughout; react positively throughout; change from positive to negative or positive to partial negative; and change from positive to alternation between positive and negative (Ryland, 1960). Second, in larvae of Bugula flabellata and B. turrita, it is possible to artificially force a switch in photic response with agents such as elevated pH, hypotonic seawater, or CuCl (Lynch, 1947). Third, in larvae of Cryptosula pallassiana, a rise in temperature decreases the photopositive phase, but the amount of illumination has no effect on the rate of change in sign of the response to light (Ryland, 1962). Furthermore, Ryland observed that pipetting larvae also results in an immediate change from positive to negative phototaxis.
Larvae of many species with phototactic responses possess pigmented epidermal structures that, on the basis of anatomical criteria, are assumed to be photoreceptors (Ryland, 1976, 1977). Not all species with larvae that orient to light have pigmented "eyespots," however (Ryland, 1960); in these species, other specialized epidermal cell types are hypothesized to be photoreceptors (Zimmer and Woollacott, 1989). To date, all pigmented ocelli of bryozoan larvae examined at the ultrastructural level have been based on a sensory cell with an elaboration of multiple cilia that are thought to be the receptoral organelles (Woollacott and Zimmer, 1972; Hughes and Woollacott, 1978, 1980; Reed et al., 1988).
We selected Bugula neritina for detailed investigation of the monoaminergic control of phototaxis in the larvae of marine bryozoans. B. neritina is cosmopolitan in temperate to tropical waters and is often abundant in specific locales at certain times of the year. Larvae of B. neritina are barrel-shaped and about 300 [[micro]meter] in diameter. General features of larval anatomy and events in metamorphosis are well documented for this species (Woollacott and Zimmer, 1971, 1972, 1978; Reed and Woollacott, 1982, 1983). The motive force for swimming is provided by the coordinated beating of the cilia of some 300 elongate coronal cells that form the extensive lateral surface of the larva. Two pigmented "eye-spots" are situated on the posterior surface, and these connect with a neural plate in the apical disc by way of an underlying equatorial nerve-muscle ring. Larvae are strongly photopositive on release and with time either change to photoneutral or alternate between photopositive and photonegative.
We report that 5HT and its immediate metabolic precursor, 5-hydroxytryptophan (5HTP), rapidly caused photopositive larvae of B. neritina to become photonegative. Antibodies to 5HT label cells with processes that are associated with the apical disc and extend orally to the equatorial nerve-muscle ring. In contrast, we show that DA and the [D.sub.2] DA receptor agonist quinpirole prolonged photopositivity in newly released larvae, and we present chromatographic data to confirm the presence of endogenous DA. We also report qualitative observations on the effects of 5HT and DA on the photic behavior of larvae from five other species of bryozoans and from seven species in four additional phyla. These results demonstrate that the responses observed in B. neritina to 5HT and DA are generalizable neither to larvae of congeners nor to a broad range of marine invertebrate larvae.
Materials and Methods
Behavior of larvae of Bugula neritina
Sexually mature colonies of Bugula neritina (Linnaeus) 1758 were collected in 1993, during February and March, from the sides of floating docks and other submerged objects in the Indian River near the Smithsonian Marine Station at Link Port, Fort Pierce, Florida. Colonies were maintained in 2-gal aquaria provided with aeration. Release of larvae was initiated by exposing colonies to sunlight following an overnight period of dark adaptation.
Phototaxis experiments were conducted in MBL artificial seawater (MBL-ASW, Cavanaugh, 1956) with the salinity adjusted to habitat level (20[per thousand]). Phototactic behavior of larvae was assessed in the presence of 5-hydroxytryptamine hydrochloride (serotonin, 5HT, Sigma); 5-hydroxy-L-tryptophan (5HTP, Sigma); 3-hydroxytyramine hydrochloride (dopamine, DA, Sigma); ([+ or -])-SKF-38393 (a [D.sub.1] DA receptor agonist, Research Biochemicals Inc.); and (-)-quinpirole hydrochloride (a selective [D.sub.2] DA receptor agonist, Research Biochemicals Inc.). Twenty milliliters of MBL-ASW served as the control in all experiments. Drugs were dissolved and diluted directly into MBL-ASW. Final working solutions were in 20-ml volumes and contained [10.sup.-5], [10.sup.-6], [10.sup.-7], or [10.sup.-8] M concentrations of the drug.
Experiments were conducted in glass Stender dishes with a capacity of 30 ml and an inside diameter of 4.5 cm. Larvae were transferred into dishes by pipetting, usually carrying over about 0.2-0.3 ml of Indian River water. It was not feasible to accurately determine the number of larvae being transferred; consequently, dishes contained unequal numbers of larvae. The number of larvae varied from 20 to 78 per dish, but 25 to 35 was the most common range of values. Pipetting did not affect the responses of larvae of B. neritina to light; this result is contrary to the observations of Ryland (1962) with larvae of another bryozoan, Cryptosula pallasiana. A 4-ft GE F400CW cool white fluorescent lamp was used as the light source. Dishes containing larvae were placed on white paper 3 in. from the light. Photon flux was measured with a LI-COR model LI-1000 light meter equipped with a detector calibrated for air and measuring between 400 and 700 nm. Irradiance measurements were made in air and ranged between 4.76 X [10.sup.15] and 5.78 X [10.sup.15] photons/s/[cm.sup.2] along the length of the tube. The gradient across the dishes was 6.69 X [10.sup.15] to 4.88 X [10.sup.15] photons/s/[cm.sup.2]. Temperature remained within 1 [degree] C over the course of individual experiments and ranged from 20 [degrees] C to 23 [degrees] C over the course of all experiments.
The number of larvae in the half of the dish that was closest to the light was counted at designated time intervals throughout an experiment. Total numbers of larvae were then counted and the data converted to percentages. A repeated-measures ANOVA was used on arcsin-transformed data to determine whether significant differences existed among treatments, and a Fisher probability least significant difference test was used to localize where these differences, if any, resided (Statview 4.0, Abacus Concepts).
Behavior of other marine invertebrate larvae
The phototactic responses of larvae of 12 additional species of marine invertebrates were evaluated qualitatively (Table I). Some of these studies were conducted at the Museum of Comparative Zoology in Cambridge, Massachusetts, and others at the Kewalo Marine Laboratory in Honolulu, Hawaii. In all cases, 5HT and DA were applied at a concentration of [10.sup.-5] M. As above, 20-ml volumes in 30-ml Stender dishes were used. A combination of fluorescent and natural light was employed, but irradiance was not quantified. Responses of larvae were gauged qualitatively.
Scanning electron microscopy
Larvae of Bugula neritina were fixed in 1% Os[O.sub.4] in seawater for 30 min, rinsed in distilled water, and dehydrated in a graded series of acetone. Specimens were critical-point dried in C[O.sub.2], gold-palladium coated, and examined with an AMR 1000 scanning electron microscope.
The immunohistochemical protocol was adapted from that of Kempf et al. (1987). Larvae were fixed for 1 h at room temperature in 4% paraformaldehyde containing 0.14 M NaCl and 0.2 M Millonig's phosphate buffer (MPB). After fixation, larvae were washed twice in MPB (10 min each). Larvae were then washed in deionized distilled water (DDW) and dehydrated through an ethanol series to xylene (30%, 50%, 80%, 95%, 100%, 100%, xylene, 10 min each), then rehydrated through the same series back to DDW. This procedure increases tissue permeability to antibodies. Larvae were then washed twice (10 min each) in 20 mM phosphate-buffered saline in 0.15 M NaCl, pH 7.3, containing 0.1% Triton X-100 and 0.1% Na[N.sub.3] (PBS+). Subsequent steps were carried out on a shaker table at 4 [degrees] C. Larvae were incubated for 4 h in a blocking medium consisting of 5% heat-inactivated goat serum in PBS+, then incubated overnight in primary antibody solution. This was a 1:325 dilution (in blocking medium) of polyclonal rabbit anti-5HT (Incstar #20080). A batch of larvae was treated overnight with blocking medium instead of primary antibody solution, as a control for specificity of immunofluorescent labeling. All larvae were then washed four times with PBS+ over a 12-h period, and incubated overnight in the secondary antibody solution. Secondary antibodies were goat anti-rabbit immunoglobulin G conjugated to fluorescein isothiocyanate (Organon-Teknica-Cappel #12121671), diluted 1:300 in blocking medium. Following treatment with secondary antibody, larvae were again washed four times with PBS+ over a 12-h period, then mounted in a Tris-buffered (pH 9.5) glycerol medium containing 4% n-propyl gallate (Giloh and Sedat, 1982). Larvae were examined and photographed under UV epi-illumination.
Chromatographic analysis of dopamine
DA content of larvae was analyzed by high-pressure liquid chromatography (HPLC) with electrochemical detection. Homogenization of larvae and extraction of catecholamines differed only in a few details from the methods of Coon and Bonar (1986). Approximately 75 [[micro]liter] of packed larvae was concentrated by gentle centrifugation and homogenized in a glass tissue grinder on ice in 1.0 ml of 0.4 N perchloric acid with 4 mM reduced glutathione, 4.7 mM EGTA, and 100 nM 3,4-dihydroxybenzylamine (DHBA, Sigma) as an internal standard. The homogenate was centrifuged at 15,000 X g for 5 min. Two 400-[[micro]liter] aliquots were transferred to 10 X 75 mm glass tubes for extraction of catecholamines over alumina (Anton and Sayre, 1962). To each tube was added 600 [[micro]liter] DDW, 50 mg alumina, and 1 ml extraction buffer (1.5 M Tris plus 50 mM [Na.sub.2] EDTA, pH 8.6). Tubes were agitated 10 min by inversion. Alumina was washed twice with DDW, transferred to microcentrifuge filter assemblies (Rainin #39-402), and catechols were eluted with 200 [[micro]liter] 0.1 N perchloric acid. This extract was injected directly into the HPLC system (standard injection volume 80 [[micro]liter]). Each extraction therefore provided enough material for two full sample injections; the remaining material from each extraction was used in smaller injections to help confirm the DA peak while varying the organic content of the mobile phase and "spiking" the chromatographic standards mix (see below). The recovery efficiency of the DHBA internal standard (64%-68%) was calculated for each extraction and used to correct the endogenous catecholamine data from that extraction.
Separation of catecholamines was accomplished by HPLC with an Alltech #28922 Adsorbosphere catecholamine column (100 X 4.6 mm, 3 [[micro]meter] C-18 reverse phase packing). The aqueous portion of the mobile phase contained 100 mM monochloroacetic acid, 1.3 mM [Na.sub.2] EDTA, and 1.3 mM sodium octyl sulfate adjusted to pH 3.00-3.05 with NaOH (85 mM final concentration). To this was added 1%-5% (v/v) acetonitrile. Flow rate was set at 1.0 ml/min.
Catecholamines were detected with an EG & G/Princeton Applied Research #400 amperometric electrochemical detector with glassy carbon working electrode set at an oxidizing potential of 700 mV against a Ag/AgCl reference electrode (Riggin and Kissinger, 1977; Krstulovic, 1982). Oxidation current range was set at 10 or 20 nA full-scale for the smaller peaks in the first part of the chromatogram, and at 50 nA for DA, which eluted later and was present at higher concentration. Detector output was low-pass filtered (1.0-s time constant) and sent to a Beckman #427 integrator which printed chromatograms and calculated peak areas and retention times.
The DA chromatogram peak was identified by comparing its retention time to that of authentic DA at various concentrations of acetonitrile (1%-5%). A standard curve of DA peak area as a function of amount injected was used to quantify DA concentration in larval homogenates. Reported values of larval DA are derived from the mean of two 80-[[micro]liter] injections, one from each extraction, run at the acetonitrile concentration (5%) that yielded best resolution of the DA peak. Dopamine concentration was expressed as picomoles of free base per microgram of protein. Protein content of the homogenate was assayed by the Coomassie blue dye-binding method (Bradford, 1976) with bovine serum albumin as the standard (Bio-Rad kit, #500-0002).
Monoamine modulation of phototactic behavior of larvae of Bugula neritina
Preliminary experiments conducted during June 1992 in Honolulu, Hawaii, established in a qualitative fashion that the addition of exogenous 5HT at [10.sup.-5] M concentration brought about an. immediate transformation from positive to negative phototaxis in newly released larvae of B. neritina. At that time, stocks of adult colonies were insufficient to provide the quantities of larvae needed for a full experimental analysis, but the response was reconfirmed in Hawaiian B. neritina during May 1993.
The effects of 5HT and, subsequently, DA on phototaxis were evaluated in detail in February and March 1993 in Fort Pierce, Florida, where a large supply of sexually mature colonies was available.
The effect of 5HT on the percentage of positively phototactic larvae was evaluated at concentrations of [10.sup.-5], [10.sup.-6], [10.sup.-7], and [10.sup.-8] M over a 140-min period [ILLUSTRATION FOR FIGURE 1A OMITTED]. In controls without applied 5HT, the percentage of larvae remaining photopositive decreased from a mean of 88% to 70% over the 140-min period. There was no significant difference from the control in experimental treatments with [10.sup.-8] M exogenous 5HT. However, the decrease in photopositivity seen after 140 min in larvae treated with [10.sup.-7], [10.sup.-6], and [10.sup.-5] M 5HT was much greater than that seen in controls (P [less than] 0.05 for [10.sup.-7] M; P [less than] 0.02 for [10.sup.-6] and [10.sup.-5] M 5HT). The change from photopositive was especially rapid at [10.sup.-6] and [10.sup.-5] M concentrations. At these concentrations, most larvae switched to photonegative within about 1 min after the addition of 5HT; in every trial at [10.sup.-5] M all larvae were photonegative after 110 min. Parallel studies of change in sign of phototaxis were conducted using 5HTP, the immediate metabolic precursor of 5HT [ILLUSTRATION FOR FIGURE 1B OMITTED]. Results of these studies documented a shift in phototaxis even more dramatic than that observed with 5HT. The decrease in percentage of positively phototactic larvae after 140 min was significantly greater at [10.sup.-5], [10.sup.-6], and [10.sup.-7] M 5HTP than in control trials (P [less than] 0.02). The response to 5HTP was also more rapid at the higher concentrations than was the response to 5HT at those same concentrations.
The effect of DA on the percentage of positively phototactic larvae was assessed at [10.sup.-5], [10.sup.-6], [10.sup.-7], and [10.sup.-8] M concentrations over a 240-min period [ILLUSTRATION FOR FIGURE 2A OMITTED]. In controls lacking exogenous DA, the percentage of larvae remaining photopositive decreased from a mean of 83% to 41% over 240 min. The addition of DA at [10.sup.-7], [10.sup.-6], and [10.sup.-5] M levels caused more larvae to remain photopositive than in the untreated controls (P [less than] 0.02). Larvae treated with [10.sup.-5] M DA did not show any appreciable loss of positive phototaxis up to 240 min after application of DA. The response of larvae of B. neritina to DA was qualitatively reconfirmed using Hawaiian material in May 1993. Application of the [D.sub.1] DA receptor agonist SKF-38393 had no significant effect on phototactic behavior of larvae of B. neritina at concentrations of [10.sup.-5] to [10.sup.-8] M, but was clearly toxic to the larvae. The [D.sub.2] DA receptor agonist quinpirole, however, mimicked the effect of DA in prolonging positive phototaxis. The percentage of photopositive larvae was significantly greater after 240 min in quinpirole treatments of [10.sup.-5] and [10.sup.-6] M than in the untreated controls (P [less than] 0.02, [ILLUSTRATION FOR FIGURE 2B OMITTED]). Application of quinpirole at [10.sup.-7] and [10.sup.-8] M produced an initial decrease in the percentage of photopositive larvae compared with the controls, but the difference was no longer significant after 240 min.
Localization of serotonin
Larvae of B. neritina have two pigmented eyespots situated in two depressions on the posterior lateral surface of the larva [ILLUSTRATION FOR FIGURE 3 OMITTED]. The lateral surfaces of the larva are formed by heavily ciliated cells of the corona, the larval locomotory organ. An apical disc complex with a central neural plate is situated at the aboral pole of the larva.
Immunolocalization of 5HT was achieved in larvae of B. neritina, but the procedure resulted in distortion of larvae and made precise determination of sites of the reaction difficult [ILLUSTRATION FOR FIGURE 4 OMITTED]. Strong 5HT-like immunoreactivity is evident, however, in the region of the equatorial nerve-muscle ring and in two tracts extending from the apical disc to this ring. These tracts are in a position occupied by a nerve-muscle tract extending from the roof of the metasomal sac and equatorial nerve-muscle ring to the neural plate in the center of the apical disc. The equatorial nerve-muscle ring underlies the two posterior lateral pigmented eyespots. No fluorescence above background was observed in control larvae in which the anti-5HT primary antibody was omitted.
Chromatographic analysis of dopamine
Reversed-phase HPLC of two alumina extracts of a homogenate of newly released larvae of B. neritina yielded chromatograms that included several peaks, including one that was identified as DA on the basis of its co-elution with authentic DA over a range of mobile-phase acetonitrile concentrations [ILLUSTRATION FOR FIGURE 5 OMITTED]. The two extractions resulted in DA values of 516 and 481 pmol/homogenate; these were averaged and divided by the total soluble protein content of the homogenate to yield a final estimate of larval DA content of 0.120 pmol/[[micro]gram] protein. Dihydroxyphenylalanine and norepinephrine may also be present in larvae of B. neritina [ILLUSTRATION FOR FIGURE 5 OMITTED]: all chromatograms showed at least a partially resolved peak corresponding to each of these monoamines, but we did not have enough material to justify reporting quantitative values.
Comparative analysis of larval phototaxis
We examined the effects of 5HT at [10.sup.-5] M concentration, and in some cases [10.sup.-5] MDA as well, on phototaxis of larvae from a number of species (Table I). Although the analysis was based exclusively on a qualitative evaluation of responses to the addition of these monoamines, we were unable to document a pattern similar to that observed in B. neritina in larvae from two congeners and three additional species of bryozoans. In a broader survey we did not detect any effects of monoamines on phototaxis of larvae of one copepod crustacean, one gastropod, two demosponges, and two ascidians. The larvae of the gastropod Phestilla sibogae, however, did present increased motility and greater positive phototaxis when exposed to [10.sup.-5] M 5HT.
Bath-application of [10.sup.-5] Mor [10.sup.-6] M 5HT rapidly induced negative phototaxis in newly released photopositive larvae of B. neritina [ILLUSTRATION FOR FIGURE 1A OMITTED]. A similar response was obtained with the same concentrations of 5 HTP, the immediate metabolic precursor of 5HT, although the onset of negative phototaxis took about 10-15 min longer with 5HTP than with 5HT [ILLUSTRATION FOR FIGURE 1B OMITTED]. This result is consistent with the notion that serotonergic cells may take up exogenous 5HTP and convert it to 5HT, thus augmenting releasable endogenous stores of that neuromodulator. In the gastropod Lymnaea stagnalis, injection of 5HT activates rhythmic shell movements; the same motor program can be obtained by injection of 5HTP, which also increases levels of 5HT and firing of serotonergic neurons (Kabotyanski et al., 1992).
Dopamine, when bath-applied in concentrations from [10.sup.-7] Mto [10.sup.-5] M, prolonged the initial period of photopositivity in larvae of B. neritina [ILLUSTRATION FOR FIGURE 2A OMITTED]. This effect was mimicked by the mammalian D2 DA receptor agonist quinpirole [ILLUSTRATION FOR FIGURE 2B OMITTED] at [10.sup.-6] M and [10.sup.-5] M. The sharp initial decrease in the percentage of photopositive larvae seen after treatment with [10.sup.-7] M quinpirole, contrasted with enhancement of phototaxis at higher concentrations, may reflect interactions with more than one class of DA receptor; no pharmacological profiles of bryozoan DA receptors are available.
Under normal laboratory conditions, larvae of B. neritina remain photopositive for 2-3 h (see controls in [ILLUSTRATION FOR FIGURE 2 OMITTED]), then make a transition to a state in which they alternate between positive and negative phototaxis (Ryland, 1960), and eventually may become photonegative (Mawatari, 1951). Lynch (1947) never observed negative phototaxis in this species under normal laboratory conditions of lighting, temperature, and salinity, but he was able to induce larvae to become photonegative by exposing them to intense light. He inferred from distributions of adult colonies in the field that most larvae in nature are probably photonegative at the time of settlement and metamorphosis.
It is possible that 5HT and DA are neurochemical effectors of an ontogenetic switch in the sign of phototaxis. Opposing peripheral cilioexcitatory and both peripheral and central cilioinhibitory roles, respectively of serotonin and dopamine, have been described in the gill epithelium of the bivalve Mytilus edulis (Catapane et al., 1978; Aiello et al., 1986). However, such mechanisms are probably not adequate to explain the aminergic control of cilia-driven phototaxis documented in this report. Because 5HT and DA exert opposite effects on the sign of phototaxis when applied homogeneously to the entire animal, it seems more likely that they modulate the interaction between photoreceptor organs and the ciliated coronal cells that are the effectors of phototaxis (Woollacott and Zimmer, 1972). Larvae of B. neritina always swim with the aboral pole directed forward, while rotating clockwise about the oral-aboral axis. In both positive and negative phototaxis the sensory feedback to the larva's locomotion should operate to equalize the light input to the two laterally situated photoreceptor organs; this would keep the oral-aboral axis parallel to the direction of the light stimulus. The crucial difference between positive and negative phototaxis is expected to be in the larva's course-correction mechanism. In positive phototaxis, asymmetric illumination of the two photoreceptor organs should result in inhibition of cilia on the more-illuminated side, or excitation of cilia on the lessilluminated side (Woollacott and Zimmer, 1972). To achieve negative phototaxis, the opposite course-correction strategy would be required: inhibition of cilia on the less-illuminated side, or excitation of cilia on the more-illuminated side.
If phototaxis is synaptically mediated by cells of the equatorial nerve-muscle ring, as has been suggested on anatomical grounds (Woollacott and Zimmer, 1972), one can propose that a functional "rewiring" of the phototaxis control system might be accomplished by monoamine neuromodulators. That is, whether a light stimulus to a photoreceptor organ excites or inhibits a given population of cilia might depend on the neuromodulatory milieu of the synapses in the phototaxis control system. In the pyloric network of the stomatogastric ganglion of the lobster Panulirus interruptus, the qualitative pattern of functional connections between identified neurons has been shown to depend critically upon the modulatory neurohormonal environment of the circuit (Johnson et al., 1995). In two instances DA actually reverses the sign of an identified mixed chemical/electrical synaptic connection, enhancing chemical inhibition and reducing electrical coupling so that the net synaptic interaction changes from excitatory to inhibitory (Johnson et al., 1993).
Although we have demonstrated clear and dramatic effects of bath-applied 5HT and DA on the photic behavior of larvae of B. neritina, participation of endogenous 5HT and DA in the control of phototaxis remains to be established. However, such roles for these amines seem quite likely. Processes of 5HT-immunoreactive cells are localized in the equatorial nerve-muscle ring [ILLUSTRATION FOR FIGURE 4 OMITTED], where they are situated to modulate the interactions between [TABULAR DATA FOR TABLE I OMITTED] the putative photoreceptoral organs and the coronal effectors of locomotion (Woollacott and Zimmer, 1972). Dopamine was not localized in the present study, but amounts of DA per microgram of total protein in newly released larvae of B. neritina are within the range reported for molluscan larvae, in which DA has been implicated in the control of settlement (Coon and Bonar, 1986; Bonar et al., 1990) and metamorphosis (Pires et al., 1992; Pires et al., 1995).
It is indeed puzzling that newly released photopositive larvae of B. stolonifera and B. turrita were unresponsive to [10.sup.-5] M bath-applied 5HT. It may be that in these species the conditions that would permit 5HT to influence phototaxis do not apply early in larval life, or at all. Resolution of this issue will require detailed neurochemical and behavioral investigations across the duration of the larval period.
This study was made possible through the kindness of M. Hadfield (Kewalo Marine Laboratory, University of Hawaii, Honolulu, Hawaii) and M. Rice (Smithsonian Marine Station at Link Port, Fort Pierce, Florida), who sponsored our work at their respective facilities. SEM analysis was conducted by E. Seling at the Museum ofComparative Zoology, Harvard University. During this project AP was a postdoctoral fellow supported by NSF Grant #DCB89-03800 to M. Hadfield. Support provided RMW by a Short-Term Visitor Award from the Smithsonian Institution and a Putnam Expedition Grant from the Museum of Comparative Zoology, Harvard University, is gratefully acknowledged. This is contribution number 428 from the Smithsonian Marine Station at Link Port.
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|Author:||Pires, Anthony; Woollacott, Robert M.|
|Publication:||The Biological Bulletin|
|Date:||Jun 1, 1997|
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