Anthony Pires

Development of serotonin-like immunoreactivity in the embryos and larvae of nudibranch mollusks with emphasis on the structure and possible function of the apical sensory organ.

This investigation provides a light and electron microscopic examination of the development of serotonin‐like immunoreactivity and structure of the apical sensory organ (ASO) in embryos and/or larvae of four nudibranch species: Berghia verrucicornis, Phestilla sibogae, Melibe leonina, and Tritonia diomedea. Serotonin‐like immunoreactivity is first expressed in somata, dendrites, and axons of a group of five distinct neurons within the ASO. These neurons extend axons into an apical neuropil, a structure that is situated centrally and immediately dorsal to the cerebral commissure. Three of these neurons possess sensory dendrites that extend through the pretrochal epithelium, each supporting two cilia at their distal ends. Later development of serotonin‐like immunoreactivity includes 1) axons from the apical neuropil that extend into each of the velar lobes; 2) neuron perikarya in the cerebral and pedal ganglia; 3) axons that extend through the cerebral commissure, cerebral‐pedal connectives, pedal commissure, and possibly the visceral loop connective; and 4) axons extending from each pedal ganglion into the larval foot. Ultrastructurally, the ASO can be seen to be composed of three lobes and an apical neuropil that is separately delineated from the cerebral commissure. Four cell types are present within the ASO: ciliary tuft cells, type I and type II parampullary neurons, and ampullary neurons. Immunofluorescence and 3,3′ diaminobenzidine tetrahydrochloride (DAB) labeling verify that the serotonergic neurons of the ASO are type I and type II parampullary neurons. The ampullary and type I parampullary neurons possess dendrites that extend through the pretrochal epithelium. These dendrites are partitioned into three bundles, one on either side of the ciliary tuft cells and a third bundle penetrating the pretrochal epithelium centrally between the ciliary tuft cells. One serotonergic type I parampullary neuron is associated with each of these bundles. Two ampullary neurons are associated with each of the lateral dendritic bundles, while the central bundle includes only one. Ultrastructural analyses of serotonergic axonal innervation arising from the ASO agree with those determined from fluorescently labeled material. The structure of the ASO and its associated serotonergic axons suggest that the serotonergic component of this structure senses environmental stimuli affecting velar function, possibly the contractility of muscle fibers in the velar lobes. Similarities and differences among the ASOs of embryos and larvae from various invertebrate phyla may provide useful data that will assist in the reconstruction of phylogenetic relationships. J. Comp. Neurol. 386:507‐528, 1997.

Catecholamines modulate metamorphosis in the opisthobranch gastropod Phestilla sibogae.

Larvae of the nudibranch Phestilla sibogae are induced to metamorphose by a factor from their adult prey, the coral Porites compressa. Levels of endogenous catecholamines increase 6 to 9 days after fertilization, when larvae become competent for metamorphosis. Six- to nine-day larvae, treated with the catecholamine precursor L-DOPA (0.01 mM for 0.5 h), were assayed for metamorphosis in response to coral inducer and for catecholamine content by high-performance liquid chromatography. L-DOPA treatment caused 20- to 50-fold increases in dopamine, with proportionally greater increases in younger larvae, so that L-DOPA-treated larvae of all ages contained similar levels of dopamine. A much smaller (about twofold) increase in norepinephrine occurred in all larvae. The treatment significantly potentiated the frequency of metamorphosis of 7- to 9-d larvae at low concentrations of inducer. In addition, L-DOPA treatment at 9 d increased aldehyde-induced fluorescence in cells that were also labeled in the controls, and revealed additional cells. However, all labeled cells were consistent with the locations of cells showing tyrosine-hydroxylase-like immunoreactivity. Catecholamines are likely to modulate metamorphosis in P. sibogae, but rising levels of catecholamines around the time of competence are insufficient alone to account for sensitivity to inducer in competent larvae.

Oxidative Breakdown Products of Catecholamines and Hydrogen Peroxide Induce Partial Metamorphosis in the Nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia)

Veliger larvae of the aeolid nudibranch Phestilla sibogae metamorphose in response to a soluble factor from their prey coral, Porites compressa. Metamorphosis begins with destruction of the velum, a ciliated structure used for swimming and feeding. Previous investigation had shown that P. sibogae larvae exposed to certain catecholamines lost the velum, but then failed to complete any subsequent steps characteristic of natural coral-induced metamorphosis. Because catecholamines oxidize rapidly in seawater, we have re-examined morphogenic effects of catecholamines using superfusion chambers that allow periodic replacement of test solutions. We report that fresh, unoxidized catecholamines do not induce velar loss, but that this morphogenic activity develops in aged, oxidized solutions of a variety of catecholamines and other catechol compounds. Evidence is presented that this activity is attributable to hydrogen peroxide, a byproduct of catechol autoxidation. Hydrogen peroxide induces velar loss at 10-4 M. The possible relationship of peroxide-induced velar loss to natural coral-induced metamorphosis is discussed.

Serotonin and Dopamine Have Opposite Effects on Phototaxis in Larvae of the Bryozoan Bugula neritina

Adult colonies of the bryozoan Bugula neritina release short-term anenteric larvae that initially are strongly photopositive. Over the course of several hours larvae lose their initial photopositivity and either become photonegative or alternate between positive and negative phototaxis. We report that newly released photopositive larvae rapidly become photonegative upon exposure to 10-6-10-5 M serotonin or its metabolic precursor, 5-hydroxytryptophan. This behavior was not observed in two congeners of B. neritina, nor in larvae of three other species of bryozoans and seven species from four additional phyla. Antibodies to serotonin label cells in the region of the equatorial nerve-muscle ring and in two tracts extending from the apical disc to this ring. In a separate series of experiments, larvae treated with dopamine (10-7-10-5 M) significantly prolonged their photopositive period. This effect was also obtained with the D2 dopamine receptor agonist, quinpirole (10-6-10-5 M). HPLC analysis determined that newly released photopositive larvae contained 0.120 pmol dopamine/ {mu}g protein. These findings implicate serotonin and dopamine as important neurochemical regulators of phototaxis in larvae of B. neritina.

Nitric Oxide Inhibits Metamorphosis in Larvae of Crepidula fornicata, the Slippershell Snail

This paper concerns the role of nitric oxide (NO) in controlling metamorphosis in the marine gastropod Crepidula fornicata. Metamorphosis was stimulated by the nitric oxide synthase (NOS) inhibitors AGH (aminoguanidine hemisulfate) and SMIS (S-methylisothiourea sulfate) at concentrations of about 100–1000 μmol l−1 and 50–200 μmol l−1, respectively. Metamorphosis was not, however, induced by the NOS inhibitor l-NAME (l-NG-nitroarginine methyl ester) at even the highest concentration tested, 500 μmol l−1. Moreover, pre-incubation with l-NAME at 20 and 80 μmol l−1 did not increase the sensitivity of competent larvae to excess K+, a potent inducer of metamorphosis in this species; we suggest that either l-NAME is ineffective in suppressing NO production in larvae of C. fornicata, or that it works only on the constitutive isoform of the enzyme. In contrast, metamorphosis was potentiated by the guanylate cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3, -a]quinoxalin-1-one) in response to a natural metamorphic inducer derived from conspecific adults. Because NO typically stimulates cGMP production through the activation of soluble guanylate cyclase, this result supports the hypothesis that NO acts as an endogenous inhibitor of metamorphosis in C. fornicata. The expression of NOS, shown by immunohistochemical techniques, was detected in the apical ganglion of young larvae but not in older larvae, further supporting the hypothesis that metamorphosis in C. fornicata is made possible by declines in the endogenous concentration of NO during development.

Transmitter contents of cells and fibers in the cephalic sensory organs of the gastropod mollusc Phestilla sibogae

While the central ganglia of gastropod molluscs have been studied extensively, relatively little is known about the organization and functions of the peripheral nervous system in these animals. In the present study, we used immunohistochemical procedures to examine the innervation of the rhinophores, oral tentacles and region around the mouth of the aeolid nudibranch, Phestilla sibogae. Serotonin-like immunoreactivity was found in an extensive network of efferent projections apparently originating from central neurons, but was not detected within any peripheral cell bodies. In contrast, large numbers of peripheral, and presumably sensory, somata exhibited reactivity to an antibody raised against tyrosine hydroxylase (the enzyme catalyzing the initial step in the conversion of tyrosine into the catecholamines). Additional tyrosine hydroxylase-like immunoreactivity was detected in afferent fibers of the peripheral cells and in several cells within the rhinophoral ganglia. The presence of serotonin, dopamine and norepinephrine in the rhinophores, tentacles and central ganglia was confirmed using high-performance liquid chromatography. Finally, FMRFamide-like immunoreactivity was detected in cells and tangles of fibers found within the rhinophore, possibly revealing glomerulus-like structures along olfactory pathways. FMRFamide-like immunoreactivity was also found in somata of the rhinophoral ganglia, in a small number of cells located in the body wall lateral to the tentacles and in what appeared to be varicose terminals of efferent projections to the periphery. Together, these results indicate several new features of the gastropod peripheral nervous system and suggest future experiments that will elucidate the function of the novel cells and innervation patterns described here.

Catecholamines in larvae and juveniles of the prosobranch gastropod, Crepidula fornicata

We investigated roles of catecholamines in metamorphosis of the prosobranch gastropod, Crepidula fornicata. Levels of DOPA, norepinephrine (NE) and dopamine (DA) were measured by high-pressure liquid chromatography (HPLC) in competent larvae and juvenile siblings that metamorphosed in response to the natural adult-derived cue or to elevated K+. Competent larvae contained 1.58 +/- 0.26 (S.E.M.) x 10(-2) pmol DOPA, 0.91 +/- 0.45 x 10(-2) pmol NE, and 0.290 +/- 0.087 pmol DA (mean values per microg total protein, n = 4 batches of larvae). Levels of DA per individual were not different between larvae and juvenile siblings; levels of NE were higher in juveniles. The tyrosine hydroxylase (TH) inhibitor alpha-methyl-DL-m-tyrosine (alpha-MMT) depleted DOPA and DA to approximately half of control values without affecting levels of NE. Depletion of DOPA and DA was accompanied by inhibition of metamorphosis in response to the natural cue but not to elevated K+. The dopamine-beta-hydroxylase inhibitor diethyldithiocarbamate (DDTC) induced high frequencies of metamorphosis at concentrations of 0.1-10 microM. In juveniles induced by 10 microM DDTC, levels of both NE and DA averaged approximately 80% of those in control larvae. Catecholamines may function as endogenous regulators of metamorphosis in C. fornicata.

Analysis of nitric oxide-cyclic guanosine monophosphate signaling during metamorphosis of the nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia)

SUMMARY The gas nitric oxide (NO), and in some cases its downstream second messenger, cyclic guanosine monophosphate (cGMP) function in different taxa to regulate the timing of life‐history transitions. Increased taxonomic sampling is required to foster conclusions about the evolution and function of NO/cGMP signaling during life‐history transitions. We report on the function and localization of NO and cGMP signaling during metamorphosis of the nudibranch Phestilla sibogae. Pharmacological manipulation of NO or cGMP production in larvae modulated responses to a natural settlement cue from the coral Porites compressa in a manner that suggest inhibitory function for NO/cGMP signaling. However, these treatments were not sufficient to induce metamorphosis in the absence of cue, a result unique to this animal. We show that induction of metamorphosis in response to the settlement cue is associated with a reduction in NO production. We documented the expression of putative NO synthase (NOS) and the production of cGMP during larval development and observed no larval cells in which NOS and cGMP were both detected. The production of cGMP in a bilaterally symmetrical group of cells fated to occupy the distal tip of rhinophores is correlated with competence to respond to the coral settlement cue. These results suggest that endogenous NO and cGMP are involved in modulating responses of P. sibogae to a natural settlement cue. We discuss these results with respect to habitat selection and larval ecology.

Inhibitors of nitric oxide synthase induce larval settlement and metamorphosis of the polychaete annelid Capitella teleta

The neurotransmitter nitric oxide (NO) has been implicated in the inhibitory control of metamorphosis of some marine gastropods, echinoderms, and ascidians. We have explored whether or not metamorphosis of metatrochophore larvae of the polychaete annelid Capitella teleta is also regulated by NO. Immunohistochemical analysis revealed a bilateral group of three large nitric oxide synthase (NOS) immunoreactive cells that lie dorsal to the pharynx and extend ventral processes toward the pharynx in the region of the dorsal pharyngeal pad. Smaller NOS-immunoreactive cells were distributed widely throughout the body but concentrated in the prostomium and pygidium. Histological analysis for NO, using the NO detector diaminofluorescein-FM, showed that NO concentration was high in the larval midgut, although diffuse amounts of NO were detected throughout the body. Inhibitors of NOS, including s-methylisothiourea sulfate, aminoguanidine hemisulfate, 7-nitroindazole, and N-methyl-L-arginine all induced settlement and metamorphosis of the Capitella larvae in a concentration-dependent manner. The NO donor nitroprusside prevented the induction of settlement and metamorphosis induced by the NOS inhibitor N-methyl-L-arginine, but did not prevent settlement and metamorphosis induced by a marine sediment extract, exogenous serotonin, or by the serotonin reuptake inhibitor fluoxetine. Pre-incubating larvae with the serotonin receptor antagonist ketanserin also inhibited settlement and metamorphosis in response to NOS inhibitors. These results suggest that endogenous production of NO maintains the larval state in C. teleta, and that endogenous serotonin stimulates metamorphosis in a way similar to that described previously for larvae from other major invertebrate phyla.

Artificial Seawater Culture of the Gastropod Crepidula fornicata for Studies of Larval Settlement and Metamorphosis

The slipper limpet, Crepidula fornicata, is a gastropod mollusc of growing importance as a research model in developmental biology and as an invasive organism. The large (>1 mm) veliger larvae of this species are well suited for neuroethological investigations of settlement and metamorphosis. In this chapter, methods are described for conditioning adult broodstock, growing microalgal food for larvae, and culturing larvae to metamorphic competence in artificial seawater. A protocol is also presented for obtaining electrophysiological recordings of ciliary arrest spikes from intact, behaving larvae, as putative neural correlates of larval settlement.