Responses of cerebral GABA-containing CBM neuron to taste stimulation with seaweed extracts in Aplysia kurodai

Aplysia kurodai distributed along Japan feeds well on Ulva pertusa but rejects Gelidium amansii with distinctive patterned movements of the jaws and radula. On the ventral side of the cerebral M cluster, four cell bodies of higher order neurons that send axons to the buccal ganglia are distributed (CBM neurons). We have previously shown that the dopaminergic CBM1 modulates basic feeding circuits in the buccal ganglia for rejection by firing at higher frequency after application of the aversive taste of seaweed such as Gelidium amansii. In the present experiments immunohistochemical techniques showed that the CBM3 exhibited gamma-aminobutyric acid (GABA)-like immunoreactivity. The CBM3 may be equivalent to the CBI-3 involved in changing the motor programs from rejection to ingestion in Aplysia californica. The responses of the CBM3 to taste stimulation of the lips with seaweed extracts were investigated by the use of calcium imaging. The calcium-sensitive dye, Calcium Green-1, was iontophoretically introduced into a cell body of the CBM3 using a microelectrode. Application of Ulva pertusa or Gelidium amansii extract induced different changes in fluorescence in the CBM3 cell body, indicating that taste of Ulva pertusa initially induced longer-lasting continuous spike responses at slightly higher frequency compared with that of Gelidium amansii. Considering a role of the CBM3 in the pattern selection, these results suggest that elongation of the initial firing response may be a major factor for the CBM3 to switch the buccal motor programs from rejection to ingestion after application of different tastes of seaweeds in Aplysia kurodai.     

Search for cerebral G cluster neurons responding to taste stimulation with seaweed in Aplysia kurodai by the use of calcium imaging

The calcium imaging method can detect the spike activities of many neurons simultaneously. In the present experiments, this method was used to search for unique neurons contributing to feeding behavior in the cerebral ganglia of Aplysia kurodai. We mainly explored the neurons whose cell bodies were located in the G cluster and the neuropile region posterior to this cluster on the ventral surface of the cerebral ganglia. When the extract of the food seaweed Ulva was applied to the tentacle-lip region, many neurons stained with a calcium-sensitive dye, Calcium Green-1, showed changes in fluorescence. Some neurons showed rhythmic responses and others showed transient responses, suggesting that these neurons may be partly involved in the feeding circuits. We also identified three motor neurons among these neurons that showed rhythmic fluorescence responses to the taste stimulation. One of them was a motor neuron shortening the anterior tentacle (ATS), and the other two were motor neurons producing lip opening-like (LO(G)) and closing-like (LC(G)) movements, respectively. Application of the Ulva extract to the tentacle-lip region induced phase-locked rhythmic firing activity in these motor neurons, suggesting that these neurons may contribute to the rhythmic patterned movements of the anterior tentacles and lips during the ingestion of seaweed.     

Preparation of High Yields of Algal Protoplasts Using Buccal Juice of Sea Hare and Commercial Cellulase

Buccal juice of the sea hare Aplysia juliana was found to degrade algal polysaccharides. The optimal enzyme composition for protoplast preparation from Undaria pinnatifida was protein at 48 μg/ml buccal juice from sea hare, 10 mg/ml cellulase Onozuka-RS, 0.4 M NaCl, 0.8 M sorbitol, 2 mg/ml dextran sulfate sodium salt, and 1 μl/ml 2-mercaptoethanol in 10 mM MES buffer (pH 6.0). Protoplasts of Eisenia bicyclis, Endarachne binghamiae (Phaeophyta), and Ulva pertusa (Chlorophyta) could also be prepared in a similar manner. Yields of these protoplasts were about 10⁷ cells per gram of fresh weight alga. Received January 26, 1998; accepted September 17, 1998.     

Two different genetic strains of stalked-Ulva (Ulvales, Chlorophyta) grow on intertidal rocky shores in Ebisujima, central Japan

Stalked‐Ulva has been recognized as an ecologic form of Ulva pertusa Kjellman with narrow and extensive stipes that grows on rocky shores with strong wave action. However, it is possible that stalked‐Ulva includes more than two taxa, because it has been shown previously that some isolates of stalked‐Ulva did not cross with U. pertusa. Therefore, further crossing tests, observations of swarmer behavior, culture studies and comparison of DNA sequences were made to clarify whether or not all stalked‐Ulva are included within U. pertusa. Crossing tests showed that stalked‐Ulva contained two different types, one that crossed with U. pertusa (Up strain) and one (N strain) that did not. The biflagellate swarmers of N strains showed negative phototaxis and were slightly larger than male and female gametes of Up strains. Thalli cultured from the swarmers of N strains released the same type of swarmers again. The N strains have an asexual life history, reproducing solely by biflagellate swarmers. Internal transcriber spacer sequence analysis indicated that these two entities are U. pertusa (Up strain) and U. fasciata Delile (N strain). In culture both strains differ morphologically from wild thalli with stipes; it seems that the two different taxa both show a petiolate morphology when growing under the same environmental conditions.     

Cerebral-buccal pathways in Aplysia californica: synaptic connections, cooperative interneuronal effects and feedback during buccal motor programs

Ingestion of seaweed by Aplysia is in part mediated by cerebral-buccal interneurons that drive rhythmic motor output from the buccal ganglia and in some cases cerebral-buccal interneurons act as members of the feeding central pattern generator. Here we document cooperative interactions between cerebral-buccal interneuron 2 and cerebral-buccal interneuron 12, characterize synaptic input to cerebral-buccal interneuron 2 and cerebral-buccal interneuron 12 from buccal peripheral nerve 2,3, describe a synaptic connection between cerebral-buccal interneuron 1 and buccal neuron B34, further characterize connections made by cerebral-buccal interneurons 2 and -12 with B34 and B61/62, and describe a novel, inhibitory connection made by cerebral-buccal interneuron 2 with a buccal neuron. When cerebral-buccal interneurons 2 and 12 were driven synchronously at low frequencies, ingestion-like buccal motor programs were elicited, and if either was driven alone, indirect synaptic input was recruited in the other cerebral-buccal interneuron. Stimulation of BN2,3 recruited both ingestion and rejection-like motor programs without firing in cerebral-buccal interneurons 2 or 12. During motor programs elicited by cerebral-buccal interneurons 2 or 12, high-voltage stimulation of BN2,3 inhibited firing in both cerebral-buccal interneurons. Our results suggest that cerebral-buccal interneurons 2 and 12 use cooperative interactions to modulate buccal motor programs, yet firing in cerebral-buccal interneurons 2 or 12 is not necessary for recruiting motor programs by buccal peripheral nerve BN2,3, even in preparations with intact cerebral-buccal pathways.     

Bioactive volatile compounds from marine algae: feeding attractants

Chemical communications play an important role in plants, fungi, and algae. Volatile organic compounds in marine algae are released into the seawater. These compounds play a role as either pheromones or allelochemicals. We observed that the turbinid gastropod Lunella coronata coreensis inhabits the intertidal zone and often grazes the green alga Ulva pertusa. Feeding tests and feeding preference studies were performed with green, brown and red algae or by using the powdered freeze-dried seaweed in agar. The snails fed on U. pertusa preferentially compared to the other marine algae, and recognized chemoreception compounds from the alga but not their structural or morphological differences. From feeding tests using artificial foods, it is suggested that the feeding attractants are in the essential oil of the alga U. pertusa.     

Spatial pattern of intertidal macroalgal assemblages associated with tidal levels

The zonation pattern of macroalgal assemblages was investigated from December 1995 to October 1996 on a semi-exposed, rocky intertidal shore at Chungdori (southwestern Korea) based on quantitative and qualitative estimates of species occurrences in 31 permanent quadrats. Variation in cover associated with tidal levels was described for 30 species (that could be discerned with the unaided eyes) including three green, five brown and 22 red algae. Macroalgae inhabiting intertidal zone exhibited distinct zonation patterns. The number of species increased with decreasing intertidal height and was independent of season. The community was dominated by five species (Gloiopeltis furcata, Gelidium divaricatum, Ulva pertusa, Sargassum horneri, Hizikia fusiformis). The intertidal assemblage at the study site can be divided into two groups based on the number of species and the population structure with the division occurring at the critical level of 34 cm above MLW (mean low water). Gloiopeltis furcata, Gelidium divaricatum, Sargassum thunbergii, Monostroma grevillei, and Myelophycus simplex were more abundant in the upper shore zone and rapidly declined in abundance with depth, relative to all other species. Gelidium amansii, Pachymeniopsis elliptica, Hizikia fusiformis, Gigartina intermedia, Laurencia sp., Chondrus ocellatus, Corallina spp. and Gigartina tenella became more dominant in the lower shore zone.     

Patterned jaw movements and the motor neuron activity during rejection of seaweed in Aplysia kurodai

Japanese Aplysia kurodai feeds well on Ulva. In the present experiments we collected several species of seaweed at a location with many animals and initially explored the preference behavior for them. The animals rejected Grateloupia, Pachydictyon, Gelidium and Laurencia with rhythmic jaw and radula movements (active rejection). The animals sometimes bit off a piece of them (biting-off response). Recording activity of muscles contributing to jaw-opening and jaw-closing in freely moving animals showed that the onset of the jaw-closing activity, which always started later than the jaw-opening activity during each cycle of ingestion of Ulva, was advanced toward that of the jaw-opening activity during each cycle of the active rejection. Semi-intact experiments also showed that application of Pachydictyon or Gelidium extract to the lip region advanced the firing onset of the jaw-closing motor neurons at the radula-retraction phase. Video analysis showed that during the Ulva response the jaws opened at the radula-protraction phase and remained half-open at the earlier radula-retraction phase, while the jaws opened similarly at the radula-protraction phase but immediately closed at the radula-retraction phase during the Pachydictyon or Gelidium response. Accepted: 8 October 1997     

Genetic examination of the type specimen of Ulva australis suggests that it was introduced to Australia

‘Ana‐aosa’, one of the most common marine green algae in Japan, was described as Ulva pertusa Kjellman in 1897 from Hakodate in northern Japan. Ulva pertusa was considered to be a temperate species, with its native distributional range restricted to northeastern Asia. Although this species has been reported from various regions outside northeastern Asia, these records have been explained as non‐indigenous populations. Recently, on the basis of genetic data and nomenclatural priority, U. pertusa was synonymized with U. australis Areschoug, a species described in 1851 from specimens collected in South Australia. Based on genetic studies, Australian populations identified as U. pertusa had been considered to have originated from Japan. However, the published genetic data on U. australis in Australia have been based only on recent collections and no historical specimens have been examined. We tested the hypothesis that native (true) U. australis is an independent species of very similar morphology to U. pertusa, but that its natural domination of shoreline habitats has been suppressed by introduced populations of U. pertusa from Asia. In the present study, we extracted DNA from the type specimen of U. australis housed in the Swedish Museum of Natural History (S) and obtained DNA sequences of the chloroplast rbcL gene and the nuclear rDNA ITS2 region. Our results show that U. australis and U. pertusa are genetically virtually identical, confirming that U. pertusa is a synonym of U. australis. This suggests that the introduction of U. australis to Australia occurred by the middle of the 19th century, when the type was collected and before there was a direct shipping route between Japan and Australia. We speculate that the introduction of U. australis to Australia occurred as a secondary introduction from non‐indigenous populations in northeastern Asia, but not directly from Japan.     

Radular mechanosensory neuron in the buccal ganglia of the terrestrial slug,Incilaria fruhstorferi

A radular mechanosensory neuron, RM, was identified in the buccal ganglia of Incilaria fruhstorferi. Fine neurites ramified bilaterally in the buccal ganglia, and main neurites entered the subradular epithelium via buccal nerve 3 (n3). When the radula was distorted by bending, RM produced an afferent spike which was preceded by an axonic spike recorded at n3. The response of RM to radular distortion was observed even in the absence of Ca²⁺, which drastically suppressed chemical synaptic interactions. Therefore, RM was concluded to be a primary radular mechanoreceptor.During rhythmic buccal motor activity induced by food or electrical stimulation of the cerebrobuccal connective, RM received excitatory input during the radular retraction phase. In the isolated buccal ganglia connected to the radula via n3s, the afferent spike, which had been evoked by electrical stimulation of the subradular epithelium, was broadened with the phasic excitatory input. Since the afferent spike was also broadened by current injection into the soma, depolarization due to the phasic input may have produced the spike broadening.Spike broadening was also observed during repetitive firing evoked by current injection. The amplitude of the excitatory postsynaptic potential in a follower neuron increased depending on the spike broadening of RM.Abbreviations CBC cerebrobuccal connective - EPSP excitatory postsynaptic potential - n1,n3 buccal nerves 1 and 3 - RBMA rhythmic buccal motor activity - RM radular mechanosensory neuron - SMT supramedian radular tensor neuron     

Comparison of agarophytes (Gelidium, Gracilaria, and Gracilariopsis) as potential resources for bioethanol production

This study explores the possibility of producing ethanol using the acid hydrolysate of three abundant agar-containing red seaweeds (agarophytes): Gelidium amansii, Gracilaria tenuistipitata, and Gracilariopsis chorda. The main component in the seaweed samples was agar, which ranged from 20 to 51 % (g g^?1 dry weight). After optimizing acid hydrolysis, 100 g of seaweed was hydrolyzed at 130 °C for 15 min with 0.2 M H₂SO₄. Then, 120 mL of a 1:2 mixture of the hydrolysate broth and basal medium was fermented in a 200-mL bottle at 30 °C for 96 h. Of the three seaweeds, G. amansii had the best ethanol yield, producing 0.23 g g^?1 of galactose or 45 % of the theoretical yield. This yield increased to 60 % after detoxification of the hydrolysate with activated carbon.     

Changes of photosynthetic performances in mature thalli of the red alga Gelidium amansii (Gelidiaceae) exposed to different salinities

Photosynthetic parameters, including net O₂ evolution, pigment content and fast chlorophyll a (Chl a) fluorescence kinetics, were studied in mature thalli of Gelidium amansii, a marine agar-producing red seaweed, exposed to different salinities (0–35?psu) for 10 days. The results showed that the net O₂ evolution at 25–32?psu was unchanged, but significantly decreased at either lower or higher salinities. Hypo-saline (15?psu and below) and hyper-saline (35?psu) conditions induced significant losses of Chl a, carotenoids and phycobiliproteins, which correlated with the decrease in the absorption flux per cross-section of fronds (ABS/CS_o). Polyphasic fluorescence transients revealed that salinities at 10 and 35?psu both caused multiple effects on photosynthetic electron transport. Algae exposed to low salinity at 10?psu showed extensive damage to the donor side of the oxygen-evolving complex (OEC), reaction centre and acceptor side of PSII. Data on net O₂ evolution showed that 35?psu salinity was more destructive than 10?psu, with the absence of marked osmotic injury to the OEC. The results of this study indicate the possibility of G. amansii cultivation in estuarine waters with 20–32?psu salinity.     

Isolation of Pollutant (Pine Needle Ash)-Responding Genes from Tissues of the Seaweed Ulva Pertusa

Genetic responses of the seaweed Ulva pertusa to pine needle ash have been compared using differential display technique. The tissue viability was assessed to evaluate the stress level with triphenyltetrazolium chloride. Total RNA, from tissues treated in seawater containing ash, was reverse transcribed and amplified by PCR with arbitrary primers. The genetic fragments responding to the stress were selectively isolated from agarose gel and sequenced with a DNA auto sequencer. According to sequence analysis, an ash-inducible gene (342 bp) and an ash-suppressed gene (1690 bp) were identified as hypothetical proteins.     

Physicochemical Studies on Sulfated Polysaccharides Extracted from Seaweeds at Various Temperatures

Sulfated polysaccharides (SPS) were extracted from three species of seaweeds of Ulvacea (Ulva pertusa, Ulva conglobata and Entromorpha prolifera) for 4 hr at various temperatures and their physicochemical properties were studied using viscometric and equilibrium sedimentation measurements in order to determine the optimum extracting condition.

Sulfated polysaccharides extracted at various temperatures from the seaweed of U. pertusa had the same physicochemical properties, while the larger molecular components of SPS was not extracted from U. conglobata and E. prolifera, at the low temperature of 30~40°C. This was confirmed by analyses of their viscosity and molecular weight and by gel filtration chromatography, in which each SPS showed two or three peaks.

The larger molecular component of SPS could be extracted at the high temperature of 80~90°C in the thermostable form.     

FORMATION OF SEROTONIN BY ISOLATED SEROTONIN-CONTAINING NEURONS AND BY ISOLATED NON-AMINE-CONTAINING NEURONS

Isolated giant serotonin-containing neurons of the cerebral ganglia of Helix pomatia were shown to produce serotonin when incubated with 5-hydroxytryptophan (5-HTP) whereas cells of the buccal ganglia, which are non-amine-containing cells did not. The rate of production was comparable to that for Ach in the isolated neurons of Aplysia. The significance of these results is discussed.     

UV‐A/BLUE LIGHT–INDUCED REACTIVATION OF SPORE GERMINATION IN UV‐B IRRADIATED ULVA PERTUSA (CHLOROPHYTA)1

Recent reduction in the ozone shield due to manufactured chlorofluorocarbons raised considerable interest in the ecological and physiological consequences of UV‐B radiation (λ=280–315 nm) in macroalgae. However, early life stages of macroalgae have received little attention in regard to their UV‐B sensitivity and UV‐B defensive mechanisms. Germination of UV‐B irradiated spores of the intertidal green alga Ulva pertusa Kjellman was significantly lower than in unexposed controls, and the degree of reduction correlated with the UV doses. After exposure to moderate levels of UV‐B irradiation, subsequent exposure to visible light caused differential germination in an irradiance‐ and wavelength‐dependent manner. Significantly higher germination was found at higher photon irradiances and in blue light compared with white and red light. The action spectrum for photoreactivation of germination in UV‐B irradiated U. pertusa spores shows a major peak at 435 nm with a smaller but significant peak at 385 nm. When exposed to December sunlight, the germination percentage of U. pertusa spores exposed to 1 h of solar radiation reached 100% regardless of the irradiation treatment conditions. After a 2‐h exposure to sunlight, however, there was complete inhibition of germination in PAR+UV‐A+UV‐B in contrast to 100% germination in PAR or PAR+UV‐A. In addition to mat‐forming characteristics that would act as a selective UV‐B filter for settled spores under the parental canopy, light‐driven repair of germination after UV‐B exposure could explain successful continuation of U. pertusa spore germination in intertidal settings possibly affected by intense solar UV‐B radiation.     

Photosynthesis and growth of Ulva ohnoi and Ulva pertusa (Ulvophyceae) under high light and high temperature conditions,and implications for green tide in Japan

The macroalga Ulva ohnoi constitutes a considerable fraction of green tides in coastal areas of Japan, but little is known about the physiological characteristics of this species. To investigate the environmental factors that promote the formation of green tides, we tested the responses of U. ohnoi and another common Japanese species, Ulva pertusa, to various levels of irradiance at different water temperatures. Because the two species are morphologically similar, we identified them using the PCR‐restriction fragment length polymorphism method. Under laboratory conditions, we evaluated the photosynthetic, dark respiration, and relative growth rate at a range of water temperatures (5 to 35°C) and photosynthetically active radiation (0 to 1000 μmol photons m^?2 s^?1). The maximum gross photosynthetic rate of U. ohnoi was larger than that of U. pertusa. The dark respiration rates revealed no significant differences among the species and temperature conditions. At 500 μmol photons m^?2 s^?1, the relative growth rate of U. ohnoi was larger than that of U. pertusa in higher temperature and the difference was the largest at 20°C. The estimated compensation irradiance and estimated saturation irradiance of U. ohnoi and U. pertusa ranged from 0.709 to 5.510 and 40.530 to 58.674 μmol photons m^?2 s^?1, which were lower than those in other intertidal green macroalgae, from 6 to 11 and 50 to 82 μmol photons m^?2 s^?1, respectively. Thus, U. ohnoi which exists as free‐floating near the water surface and accumulating inside the green tide can survive extensively in the water column of the intertidal zone, furthermore, the species can maintain rapid growth in this situation. Therefore, as a result of this study, it is suggested that the ecological success of U. ohnoi in shallow waters such as the tidal flats, estuarine, and coasts of the inner bay in comparison with U. pertusa.     

Characterization of Chlorophyll–protein Complexes Isolated from Two Marine Green Algae, Bryopsis maxima and Ulva pertusa, Growing in the Intertidal Zone

Three Chl–protein complexes were isolated from thylakoid membranes of Bryopsis maxima and Ulva pertusa, marine green algae that inhabit the intertidal zone of the Pacific Ocean off the eastern coast of Japan by dodecyl-β-d-maltoside polyacrylamide gel electrophoresis. The slowest-moving fractions showed low Chl a/b and Chl/P-700 ratios, indicating that this fraction corresponds to complexes in PS I, which is large in both algae. The intermediate and fastest-moving fractions showed the traits of PS II complexes, with some associated Chl a/b–protein complexes and LHC II, respectively. The spectral properties of the separated Chl–proteins were also determined. The absorption spectra showed a shallow shoulder at 540 nm derived from siphonaxanthin in Bryopsis maxima, but not in Ulva pertusa. The 77 K emission spectra showed a single peak in Bryopsis maxima and two peaks in Ulva pertusa. Besides the excitation spectra indicated that the excitation energy transfer to the PS I complexes differed quite a lot higher plants. This suggested that the mechanisms of energy transfer in both of these algae differ from those of higher plants. Considering the light environment of this coastal area, the large size of the antennae of PS I complexes implies that the antennae are arranged so as to balance light absorption between the two photosystems. In addition, we discuss the relationships among the photosystem stoichiometry, the energy transfer, and the distribution between the two photosystems.     

Proteomics reveals selective regulation of proteins in response to memory-related serotonin stimulation in Aplysia californica ganglia

The marine mollusk Aplysia californica (Aplysia) is a powerful model for learning and memory due to its minimalistic nervous system. Key proteins, identified to be regulated by the neurotransmitter serotonin in Aplysia, have been successfully translated to mammalian models of learning and memory. Based upon a recently published large‐scale analysis of Aplysia proteomic data, the current study investigated the regulation of protein levels 24 and 48 h after treatment with serotonin in Aplysia ganglia using a 2‐D gel electrophoresis approach. Protein spots were quantified and protein‐level changes of selected proteins were verified by Western blotting. Among those were Rab GDP dissociation inhibitor alpha (RabGDIα), synaptotagmin‐1 and deleted in azoospermia‐associated protein (DAZAP‐1) in cerebral ganglia, calreticulin, RabGDIα, DAZAP‐1, heterogeneous nuclear ribonucleoprotein F (hnRNPF), RACK‐1 and actin‐depolymerizing factor (ADF) in pleural ganglia and DAZAP‐1, hnRNPF and ADF in pedal ganglia. Protein identity of the majority of spots was confirmed by a gel‐based mass spectrometrical method (FT‐MS). Taken together, protein‐level changes induced by the learning‐related neurotransmitter serotonin in Aplysia ganglia are described and a role for the abovementioned proteins in synaptic plasticity is proposed.     

A simple method to preserve algal spores of <Emphasis Type="Italic">Ulva</Emphasis> spp. in cold storage with ampicillin

Preservation of algal spores of the green seaweed Ulva fasciata and U. pertusa was enhanced by the addition of ampicillin in f/2 medium at 4°C. The viability of preserved spores was determined by a spore germination assay at various time intervals. The germination rate of U. fasciata remained at 5% to 38% for the first five days, dropping to 1% to 6% on the 10th day of storage with various preservation treatments without ampicillin at 4°C during parameter-selecting experiments. In f/2 medium, 53% of U. fasciata spores were still viable on day 5 and 23% on day 10 at 4°C. By adding 100 μg mL⁻¹ ampicillin to f/2 medium, 90% of the spores were viable at day 40 and 61% after 100 days of storage at 4°C. Spores of U. pertusa had lower preservation rates, with viabilities of 70% at day 40 and 32% at day 100. Algal spore preservation was heavily dependent on the bacterial contamination and subsequent degradation in stock solutions. Handling editor: L. Naselli-Flores