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Anemonia sulcata may be a source of marine natural products (MNPs) due to the antioxidant and antitumor activity of its crude homogenates shown in vitro in colon cancer cells. A bioguided chromatographic fractionation assay of crude Anemonia sulcata homogenates with and without its symbiont Symbiodinium was performed to characterize their bioactive composition and further determine their biological potential for the management of colorectal cancer (CRC). The 20% fractions retained the in vitro antioxidant activity previously reported for homogenates. As such, activation of antioxidant and detoxifying enzymes was also evaluated. The 40% fractions showed the greatest antiproliferative activity in T84 cells, synergistic effects with 5-fluoruracil and oxaliplatin, overexpression of apoptosis-related proteins, cytotoxicity on tumorspheres, and antiangiogenic activity. The predominantly polar lipids and toxins tentatively identified in the 20% and 40% fractions could be related to their biological activity in colon cancer cells although further characterizations of the active fractions are necessary to isolate and purify the bioactive compounds.

Simple Summary: Colorectal cancer is one of the most frequent types of cancer in the population. Recently, invertebrate marine animals have been investigated for the presence of natural products which can damage tumor cells, prevent their spread to other tissues or avoid cancer develop. We analyzed the anemone Anemonia sulcata with and without the presence of its microalgal symbiont (Symbiodinium) as a source of bioactive molecules for the colorectal cancer therapy and prevention. Colon cancer tumor cells were exposed to Anemone extracts observing a remarkable cell death and a great antioxidant capacity. These preliminary results support that Anemonia sulcata could be a source of bioactive compounds against colorectal cancer and that the absence of its symbiont may enhance these properties. Further studies will be necessary to define the bioactive compounds of Anemonia sulcata and their mechanisms of action. Abstract: Recently, invertebrate marine species have been investigated for the presence of natural products with antitumor activity. We analyzed the invertebrate Anemonia sulcata with (W) and without (W/O) the presence of its microalgal symbiont Symbiodinium as a source of bioactive compounds that may be applied in the therapy and/or prevention of colorectal cancer (CRC). Animals were mechanically homogenized and subjected to ethanolic extraction. The proximate composition and fatty acid profile were determined. In addition, an in vitro digestion was performed to study the potentially dialyzable fraction. The antioxidant and antitumor activity of the samples and the digestion products were analyzed in CRC cells in vitro. Our results show a high concentration of polyunsaturated fatty acid in the anemone and a great antioxidant capacity, which demonstrated the ability to prevent cell death and a high antitumor activity of the crude homogenates against CRC cells and multicellular tumor spheroids, especially W/O symbiont. These preliminary results support that Anemonia sulcata could be a source of bioactive compounds with antioxidant and antitumor potential against CRC and that the absence of its symbiont may enhance these properties. Further studies will be necessary to define the bioactive compounds of Anemonia sulcata and their mechanisms of action., International Excellence Campus of the Sea (CEI.MAR) CEIJ-007 CEIJ-0012

Marine-derived fungi are an important source of many valuable compounds with original structures and diverse physico-chemical properties. In this work, the metabolomic profile of a strain of Penicillium brevicompactum, recovered from a snakelocks sea anemone (Anemonia sulcata), was investigated through the parallel application of LC-ESI-HRMS, GC-MS, and NMR. Our strategy allowed the identification of mycophenolic acid, brevianamide A, and several compounds belonging to the thiosilvatins. Among the latter, five products are reported for the first time in this species. The main product of this series, cis-bis(methylthio)silvatin, was also tested for antiproliferative activity on both cancer and non-tumoral colon cell lines.

Marine-derived fungi are an important source of many valuable compounds with original structures and diverse physico-chemical properties. In this work, the metabolomic profile of a strain of Penicillium brevicompactum, recovered from a snakelocks sea anemone (Anemonia sulcata), was investigated through the parallel application of LC-ESI-HRMS, GC-MS, and NMR. Our strategy allowed the identification of mycophenolic acid, brevianamide A, and several compounds belonging to the thiosilvatins. Among the latter, five products are reported for the first time in this species. The main product of this series, cis-bis(methylthio)silvatin, was also tested for antiproliferative activity on both cancer and non-tumoral colon cell lines.

Marine benthic organisms can be used as indicators of the quality of environmental status and as monitoring tools to detect natural or anthropogenic perturbations. In temperate waters, metabolic and biochemical responses may be governed by physiological changes driven by seasonal factors. Gathering baseline information on the mechanisms underlying seasonal acclimation patterns is therefore a critical step towards the understanding of the physiological responses of biological indicators. In poikilothermic metazoans, the production of regulatory metabolic enzymes can be used as tools for deciphering the acclimation potential. The aim of this study was to characterize the natural seasonal variability in biometric traits and enzymatic biomarkers over a 12-month period in the sea anemone, Anemonia sulcata (Anthozoa, Cnidaria), from two areas with different environmental and anthropic impacts. Seasonality and site factors affected enzymatic kinetics at tentacle levels, while seasonality, site and biometrical characteristics modulated the same enzymes in the body column of A. sulcata. The results showed a decrease in enzymatic activity in summer and an increased synthesis of enzymes in the late fall and winter months. The interaction between biometric features and temperature appeared significant for mesolittoral sea anemones but not for infralittoral specimens. This study showed seasonal patterns of variations of biochemical responses in A. sulcata, suggesting that this species, an abundant member of shallow rocky bottom communities in temperate European seas, may represent a sensitive bio-indicator organism worth considering for further ecological studies on climate change as well as for environmental monitoring programs.

The present paper reports for the first time the bioaccessibility of arsenic species in Anemonia sulcata and its metabolization in the human body after the ingestion of this seafood, a delicacy highly appreciated in the south of Spain. Speciation in tissue extracts and human fluids was carried out by anion/cation exchange chromatography with inductively coupled plasma mass spectrometry ((IC)-ICP-MS). Results obtained show that about 54% of the arsenic present in the anemones is lost after cooking, around 95% of total arsenic in cooked anemones is bioaccessible and 85% of the amount ingested is eliminated from the body by urine in 90 h. The relative abundance of As in urine varies along the time, but after the first 10 h follows the order arsenobetaine (AB) ˜ DMAV (dimethylarsinate) > arsenocholine (AsC) > tetra-methyl-arsonium (TETRA) > trimethyl-arsine oxide (TMAO) > MAV (monomethylarsonate) > iAsV > dimethylarsenoethanol (DMAE), while the major species in cooked anemones is AB followed by DMAV > dimethylmonothioarsinic acid (DMASV) > AsC > TETRA > TMAO > iAsV > glycerylphosphorylarsenocholine(GPAsC) > MAV > DMA. After 30 min from the ingestion, DMAV is the most abundant in human serum, followed by AB, MAV and AsC. Finally, the use of organic mass spectrometry allowed the standardless identification of AsC, GPAsC, DMASV and DMAE., The authors thank the Spanish Ministry of Science and Innovation project CTM2009-12858-C02-01 and the projects P08-FQM-03554 and P09-FQM-4659 from Regional Ministry of Economy, Innovation, Science and Employment (Andalusian Government) for funding. The authors also thank Dr. Vicenta Devesa and Dinoraz Velez (Instituto de Agroquimica y Tecnologia de Alimentos (IATA-CSIC), Valencia, Spain) and Dr. Riansares Munoz (Universidad Complutense de Madrid) for the kind donation of TMAO, TETRA and DMAS. M.A. Garcia Sevillano thanks the Ministerio de Educacion for a PhD scholarship.

El arsénico ha sido considerado históricamente como un veneno para los humanos, aunque sus aplicaciones han sido y son muy numerosas, y determinadas especies han resultado ser inocuas o con un mecanismo de acción tóxica desconocido. Su uso generalizado en pinturas, en la metalurgia o en la agricultura, ha provocado que este elemento se encuentre en todos los compartimientos ambientales, a veces debido también a su naturaleza geogénica. Además, aunque el incremento de normativas que regulan su uso a partir de los años 70 ha reducido sustancialmente su presencia, su estudio es un tema de gran interés en la actualidad, debido al carácter tóxico de muchas de las especies de este elemento y al problema de salud ambiental que genera en algunos países como Bangladesh. En la actualidad se sabe además que los diferentes estados de oxidación y complejos o estructuras moleculares en los que este elemento puede encontrarse presentan diferente carácter tóxico y capacidad de bioacumulación, por lo que resulta imprescindible disponer de metodologías analíticas capaces de determinar estas especies por separado, a la vez que identificar nuevas especies del elemento, y conocer su mecanismo de acción en organismos modelo y finalmente en el hombre. Teniendo en cuenta que los alimentos constituyen una importante interfase entre el medio ambiente y la salud, así como una frecuente vía de exposición, esta Tesis se ha centrado en el estudio de alimentos de origen marino, en los que habitualmente las concentraciones de arsénico suelen ser elevadas, así como en el análisis de metabolitos en organismos modelo y en el hombre. Para ello, se han seleccionado las anémonas de mar (Anemonia sulcata), de elevado consumo en Andalucía, especialmente Huelva y Cádiz, y sobre la que no existen estudios previos. En esta Tesis se ha estudiado la variación de la concentración total de arsénico durante el cocinado, la interconversión de especies durante el proceso y su bioaccesibilidad, a fin de evaluar la posible repercusión que su consumo pudiera tener en la salud del hombre. Para ello, la especiación de arsénico se ha llevado a cabo mediante el uso de técnicas analíticas de extracción tradicionales, como la extracción sólido-líquido, y otras más novedosas, como la extracción mediante sonda de ultrasonidos y microondas focalizado, desarrollando asimismo estudios de estabilidad de las especies durante su análisis. Por otra parte, el uso combinado de sistemas cromatográficos complementarios u ortogonales ha permitido la resolución de un elevado número de especies de arsénico que pueden ser cuantificadas a muy bajos niveles de concentración mediante acoplamiento en línea a un plasma de acoplamiento inductivo con detector de masas (ICP-MS). Debido a la presencia de especies de arsénico para las que no se disponía de patrones comerciales, la metodología propuesta en esta Tesis se basa en el uso de una segunda dimensión para la detección mediante espectrometría de masas orgánicas, que ha permitido la identificación por primera vez de un compuesto de arsénico tiolado en un alimento, y de un derivado de la arsenocolina en un organismo no expuesto previamente a ésta. Finalmente, este estudio comprende el análisis de metabolitos de arsénico en el hombre mediante el análisis de fluidos biológicos (sangre y orina) en voluntarios que previamente consumieron este alimento marino, así como experiencias de exposición controlada a arsénico en el organismo modelo (Mus musculus). Para este último estudio se ha llevado a cabo un análisis metabolómico basado en el uso de la infusión directa a un espectrómetro de masas de triple cuadrupolo tiempo de vuelo (QqQ-TOF), análisis quimiométrico utilizando la intensidad de los iones en el espectro como descriptores, e identificación de metabolitos por espectrometría de masas en tándem. Algunos de estos metabolitos han sido cuantificados mediante cromatografía de gases con detector de masas utilizando patrones comerciales, permitiendo todo ello relacionar la exposición a arsénico con importantes ciclos como el del metabolismo de la energía, aminoácidos, colina, metionina y degradación de fosfolípidos de la membrana celular.

Red fluorescent proteins (RFPs) attract particular attention from both scientific and practical points of view. They have a great potential to be used as in vivo markers in multicolour labelling and FRET-based applications. Since long-wave light penetrates tissue more easily, far-red FPs provide an excellent tool for whole-body imaging. Besides the discovery of naturally occurring RFPs, they can also be generated by mutagenesis of non-fluorescent chromoproteins. However, the key residues responsible for a bright red FP have to be identified. Therefore, we investigated both non-fluorescent and high fluorescent GFP-like proteins from Anemonia sulcata var. rufescens. In the course of further development of fluorescent proteins, a new class of probes opened up advances in dynamic cellular imaging. These optical highlighters are able to modify their fluorescence emission colour and intensity, respectively, due to wavelength-dependent illumination. The reversible on- and off-switching between a fluorescent and a non-fluorescent chromophore state is characteristic for a specific class of optical highlighters. A cis-trans isomerization of the chromophore was revealed as key event in the switching process in recent studies concerning the molecular switching mechanism of photoswitchable fluorescent proteins (PSFPs). A promising area of application for PSFPs is their usage in high resolution fluorescence microscopy. Due to the increasing demand for novel PSFPs, we investigated various red FPs from Anthozoa to obtain a new member of this class of optical highlighters. The widespread distribution of GFP-like proteins in zooxanthellate and azooxanthellate cnidarians and their impressive colour diversity indicates different specific biological functions, which are still not completely understood. For this reason, field studies concerning two different Anemonia species in combination with investigation of host pigmentation and several biomarkers were performed during this thesis.

The present work identifies the decapod crustaceans that associate with Anemonia sulcata on the southwestern Atlantic coast of Portugal and characterises their host use pattern. It determines whether the anemone is monopolised by any species, resulting in the exclusion of conspecifics or other decapods and, under laboratory conditions, it evaluates the degree of association between each species and A. sulcata. From all sampled anemones, 79% harboured at least 1 decapod crustacean, with the majority displaying either one or two specimens (32 and 24%, respectively). The most abundant species were the shrimp Periclimenes sagittifer and the crab Inachus phalangium (representing 36 and 31% of collected specimens, respectively), which displayed lasting associations and were commonly recorded among the tentacles of the host. The species Eualus occultus, E. complex cranchii, Clibanarius erythropus, Maja brachydactyla, Pilumnus hirtellus and Polybius (Necora) puber displayed short-term associations, were mainly present on the substratum near the base, and avoided the tentacles of A. sulcata. Periclimenes sagittifer and I. phalangium were only recorded alone or in heterosexual pairs, appearing to efficiently defend their host against conspecifics. The majority of recorded species only seem to temporarily associate with A. sulcata, in order to seek protection from predators when other shelters are unavailable., El presente trabajo identifica los crustáceos decápodos que viven asociados a la anémona Anemonia sulcata en la costa atlántica sudoccidental de Portugal y caracteriza las pautas de utilización de la especie hospedadora. Verifica si la anémona es monopolizada por alguna especie, resultando en la exclusión de conespecíficos u otros decápodos, y evalúa en condiciones de laboratorio el grado de asociación entre cada especie y A. sulcata. A partir de todas las anémonas muestreadas, el 79% ospedó como mínimo una especie de crustáceo decápodo, la mayor parte de ellas con uno o dos individuos (32 y 24% respectivamente). Las especies más abundantes fueron el camarón carideo Periclimenes sagittifer y el cangrejo Inachus phalangium (representando respectivamente el 36% y 31% de los individuos recolectados). Estas especies mostraron asociaciones duraderas y fueron muestreadas comunmente entre los tentáculos del huesped. Las especies Eualus occultus, E. complejo cranchii, Clibanarius erythropus, Maja brachydactyla, Pilumnus hirtellus y Polybius (Necora) puber mostraron asociaciones no duraderas, y estuvieron principalmente presentes sobre el sustrato cerca de la base de la anémona, evitando los tentáculos. Periclimenes sagittifer e I. phalangium solamente se hallaron sólos o en pares heterosexuales, y mostraron defender eficientemente su huésped contra conespecíficos. La mayoría de especies halladas sólo parecen asociarse temporalmente con A. sulcata para buscar protección contra predadores cuando otros refugios no están disponibles.

Despite their similar fold topologies, anthozoan fluorescent proteins (FPs) can exhibit widely different optical properties, arising either from chemical modification of the chromophore itself or from specific interactions of the chromophore with the surrounding protein moiety. Here we present a structural and spectroscopic investigation of the green FP asFP499 from the sea anemone Anemonia sulcata var. rufescens to explore the effects of the protein environment on the chromophore. The optical absorption and fluorescence spectra reveal two discrete species populated in significant proportions over a wide pH range. Moreover, multiple protonation reactions are evident from the observed pH-dependent spectral changes. The x-ray structure of asFP499, determined by molecular replacement at a resolution of 1.85 A, shows the typical beta-barrel fold of the green FP from Aequorea victoria (avGFP). In its center, the chromophore, formed from the tripeptide Gln(63)-Tyr(64)-Gly(65), is tightly held by multiple hydrogen bonds in a polar cage that is structurally quite dissimilar to that of avGFP. The x-ray structure provides interesting clues as to how the spectroscopic properties are fine tuned by the chromophore environment.

In dieser Arbeit wurde eine neue Methode zur simultanen Isolierung biologisch aktiver Inhaltsstoffe aus der Seeanemone Anemonia sulcata entwickelt, welche auf säulenchromatographischen Verfahren basiert (Ionenaustausch-, Gelfiltrations- und Adsorptionschroma-tographie). Bei den isolierten Substanzen handelt es sich um Neurotoxine mit Wirkung auf spannungsabhängige Natriumkanäle, Kaliumkanalblocker und polyvalente Proteinaseinhibitoren. Besonderes Augenmerk lag auf der Wirkung der isolierten Substanzen auf die menschliche Blutgerinnung. Ein Großteil der Wirkungen kann dabei den polyvalenten Proteinaseinhibitoren zugeordnet werden, wie z. B. die ausgeprägte aPTT-Verlängerung durch Hemmung der Gerinnungsfaktoren IX, XI, XII und des Kallikreins oder die antifibrinolytische Wirkung durch Plasmin- und t-PA-Hemmung sowie durch Steigerung der PAI-Aktivität. Auch die beobachtete Verminderung der Plättchenaggregation ist vermutlich auf die Proteinaseinhibitoren zurückzuführen. Allerdings konnten auch Einflüsse auf die Blutgerinnung festgestellt werden, die nicht mit den Proteinaseinhibitoren in Verbindung gebracht werden konnten, so die Hemmung von Gerinnungsfaktor VIII und die Erhöhung der t-PA-Aktivität, welche der antifibrinolytischen Wirkung der Proteinaseinhibitoren entgegengerichtet ist.

Evidence for the existence of chemical defence in the echiuran Bonellia viridis is provided by investigating the palatability of Bonellia tissues to the shrimp Palaemon elegans, the teleosts Oblada melanura and Mugil labeo, and the anthozoan Anemonia sulcata. Bonellia tissues are shown to be highly distasteful to the shrimp. The results with the teleosts and the anthozoan are less clear‐cut though Bonellia extracts are shown to be distasteful also to these species.

The basic polypeptide ATX II (MW 4,770) isolated from the sea anemone Anemonia sulcata evokes a pronounced and dose-dependent positive inotropic effect in different mammalian heart preparations. The mechanism of this effect is so far unknown. (a) Investigations on isolated guinea pig atria indicate that changes of the steady state cellular Na, K and Ca concentrations cannot account for the positive inotropic effect. (b) An increase of the surface pressure of phospholipid monolayers was observed only at cardiotoxic ATX II concentrations. However, the 45Ca binding to phosphatidylserine, as the essential Ca-binding phospholipid, was not changed even at cardiotoxic ATX II concentrations. (c) Neither the enzymatic activity nor the ouabain inhibition kinetic of an isolated Na/K-ATPase preparation was affected by ATX II. (d) In intact electrically stimulated (1 Hz) guinea pig atria the binding of [3H]ouabain increases by about 50% at a positive inotropic ATX II concentration. The results suggest that the positive inotropic effect of ATX II is not caused by an unspecific membrane damaging action or by a direct interaction with the Na/K-ATPase. The increased binding of [3H]ouabain to intact heart muscles indirectly reflects an increased pump activity of the Na/K-ATPase, which is caused by an elevated Na transient due to the electrophysiologically well-established mechanism of the ATX II action on fast Na channel, i.e., delayed inactivation of the fast Na flux. However, the exact mechanism of the ATX II induced positive inotropic effect remains unknown.

The effects of Anemonia sulcata toxin (ATX II) on action potentials and membrane currents were studied in single myocytes isolated from guinea-pig or bovine ventricles. Addition of ATX II (2-20 nM) prolonged the action potential duration without a significant change in resting membrane potential. Concentrations of 40 nM-ATX II or more induced after-depolarizations and triggered automaticity. The effects were reversible after washing or upon addition of 60 microM-tetrodotoxin (TTX). 5 mM-Ni did not modify the effects. The single patch-electrode voltage-clamp technique of Hamill, Marty, Neher, Sakmann & Sigworth (1981) was applied to record membrane currents in response to 8.4 S long depolarizations starting from a holding potential of -90 mV. Currents flowing later than 5 ms after the depolarizing step were analysed. The fast events could not be considered because of insufficient voltage homogeneity. After 2 min of exposure to ATX II (20 nM) the changes in net membrane currents were measured. The difference between the currents in the presence of ATX II and during control was defined as the 'ATX-II-induced current' (iATX). After 4 min of wash iATX disappeared. Within 10 S of exposure to 60 microM-TTX, iATX was blocked completely. At potentials positive to -60 mV, iATX was inwardly directed and decayed slowly but incompletely during the 8.4 S long depolarizing pulse. The rate of decay was faster during clamp pulses to more positive potentials. A high amplitude noise was superimposed on the current trace; its amplitude decreased with more positive potentials. We analysed the voltage dependence of iATX with 'isochronous' current-voltage relations. The 0.1 S isochrone of iATX was characterized by a 'threshold' for negative currents at -60 mV, a branch with a negative slope (k = -7 mV, potential of half-maximal activation (V0.5) = -38 mV, bovine cells) leading to a maximum inward current at -20 mV, and an ascending branch which led to an apparent reversal potential (Erev) around +40 mV. The values measured in guinea-pig myocytes were similar though not identical (k = -5.5 mV, V0.5 = -30 mV, maximum of inward current at -5 mV, Erev = +50 mV). Erev shifted to less positive potentials in later isochrones. Holding the membrane at -45 mV prevented the induction of extra current by ATX II. When the holding potential was then changed to -85 mV, iATX developed within some 2 min. Returning the holding potential to -45 mV blocked iATX with a similar slow time course.(ABSTRACT TRUNCATED AT 400 WORDS)

Cardiac sodium channels, modified by Anemonia sulcata toxin II, have been analyzed by the patch-clamp method. The open state of the modified sodium channels proved to be prolonged highly significantly and reopening from a closed state denoted c*-state frequently occurred, interrupted by silent periods, denoted i*-state. Activation from the c*-state was apparently not affected by toxin action, whereas activation from the i*-state was markedly prolonged. Upon higher depolarizations toxin-induced sodium channels disappeared and this behaviour has been attributed to dissociation of the toxin from the channel by use of a special pulse-protocol. The onset of the toxin effect on the action potential proved to depend on stimulation, and it is concluded that the toxin binds preferentially to the open (o)-state. Taking together the results, a kinetic scheme is suggested for action of the toxin on the cardiac sodium channel.

[Grâce a des experiences utilisant des techniques "in vitro", ont ete eleves et decrits pour la premiere fois six stades naupliens et deux stades copepodites de Paranthessius anemoniae Claus. Les methodes employees comprenaient l'aeration des elevages larvaires et l'addition a ceux-ci d'algues unicellulaires., Grâce a des experiences utilisant des techniques "in vitro", ont ete eleves et decrits pour la premiere fois six stades naupliens et deux stades copepodites de Paranthessius anemoniae Claus. Les methodes employees comprenaient l'aeration des elevages larvaires et l'addition a ceux-ci d'algues unicellulaires.]

Toxin III (ATX III) of the sea anemone (Anemonia sulcata) is a polypeptide containing 27 amino acid residues. It has no sequence similarity with other toxins (ATX I and II) from the same species, or with scorpion toxins, although they apparently act in a similar manner by prolonging action potentials. The specificity of ATX III antibodies was characterized using ATX III, ATX I, native and chemically modified ATX II, and scorpion α-toxins. The results obtained suggest that a regior of ATX III, partially or totally overlapping the pharmacological site shared with ATX I and ATX II, is immunogenic. It includes a guanidino and at least two carboxylate groups. The corresponding region is not immunogenic in ATX I and ATX II. Anti-(ATX III) antibodies recognize the similar regions of ATX I and ATX II and apparently do not recognize scorpion toxins.

SUMMARY Tissues of Elysia viridis contain a ‘factor’ which stimulates release of fixed 14C from isolated Codium chloroplasts and also from the symbiotic algae of Anemonia sulcata. This ‘factor’ appears to be thermolabile, non-particulate, and only occurs in effective amounts in the chloroplast-containing tissues of the animal. So far, attempts to mimic the action of Elysia homogenates by varying the pH of the medium, or by adding Codium homogenates, fructose 1,6 di-phosphatase or‘2C-glucose to it, have failed.

Feeding can be initiated in Anemonia sulcata by mechanical, chemical or electrical stimulation of the tentacles provided the stimulus sets up sufficiently prolonged excitation. Owing to rapid adaptation, mechanical stimuli rarely set up enduring excitation and inert objects are therefore usually rejected. Chemical stimuli set up prolonged excitation and food objects are therefore usually accepted. A series of electrical stimuli can produce rejection or feeding according to whether it is brief or prolonged. The sensitivity of the tentacles varies greatly to different foods. It is greatest to animal foods. There is great sensitivity to certain kinds of mucus. The active substances of natural foods are closely associated with protein. They fail to pass through a membrane which retains colloids. A feeding reaction can be obtained to food substances which appear to be insoluble in water. Though the active substances of many natural foods are not in free solution, soluble derivatives of proteins, such as peptones and amino acids cause a feeding reaction. With pure proteins, the response of the cnidae is diminished. With amino acids, there is no cnida response. From this it follows that increased mechanical contact due to cnida discharge is not essential for excitation of the feeding response. Fat, such as tristearin, and ethereal extracts of food are without effect. Alcoholic Soxhlet extraction of food yields a substance.which causes the food reaction. Carbohydrates are without effect except in the case of glycogen, the action of which may be due to impurities. The lower fatty acids, quinine and bile salts produce a withering contraction of the tentacles which differs from the response of the latter to food. The effect can be produced by prolonged electrical excitation at a higher frequency than that required for the feeding response and is therefore probably due to excessive stimulation. The mouth responds to a greater variety of chemical stimuli than the tentacles. The relative sensitivity of - these organs to different chemical stimuli is not the same, but for most agents the mouth is the more sensitive. Quinine, histamine and 10% bile salts excite the musculature of the mouth directly. The range of chemical sensitivity in different coelenterates is discussed.

1. The nature of the stimulus which causes discharge of the cnidae (nematocysts and spirocysts) in the tentacles of Anemonia sulcata has been studied. 2. The cnidoblasts behave as independent effectors. Contact with solid food or electric stimuli cause only local discharge. Repetitive electric stimulation at various frequencies causes spread of excitation far down the nerve net of the tentacle but the cnida discharge remains localized under the stimulating electrode. There is no physiological evidence for any connexion ofcnidoblasts with the nervous system. 3. The stimulus to the cnidoblast is primarily due to direct mechanical contact. Discharge is easily effected by solid food. It can also be effected by inert solids provided the mechanical stimulus is sufficiently intense. 4. The normal stimulus to the cnidoblast is not a directly chemical one. Most food solutions and solutions of food derivatives do not cause a discharge. Some strong food solutions cause a few scattered cnidae to discharge. This differs from the intense local discharge to solid food. 5. Immersion in solutions of certain surface-active substances, such as the lower fatty acids, bile salts and saponin, causes an immediate general discharge of all the cnidae. This differs from the normal discharge to solid food. 6. Although natural food solutions do not normally cause a discharge, the cnidae are easily discharged by contact when they are present. That is, food solutions sensitize the cnidoblasts to mechanical stimuli. 7. The sensitizing substance is not protein. But it is strongly adsorbed on to protein. It cannot be extracted with ethyl ether. But it can be extracted with ethyl alcohol or acetone. Alcohol extracts of food are very active in sensitizing the cnidoblasts to mechanical disturbance. 8. Water insoluble foods can sensitize cnidoblasts with which they come in contact. Sensitization by contact is almost instantaneous. It is suggested that normally sensitization is due to some surface active lipoid directly transferred to the cnidoblast by contact.

Anemonia sulcata resorbierte und akkumulierte tritiummarkierte l-Aminosauren aus Meerwasser, welche diesem in naturlicher Konzentration zugesetzt worden waren (70–700 nMol/l). Die Aufnahme erfolgte nahezu ausschlieslich ektodermal; der uberwiegende Teil der aufgenommenen und dann eingebauten Aminosauren befand sich auch nach langerer Inkubation im Ektoderm. Diese Tatsache ist auf unterschiedliche cytologische Differenzierungen von Ento- und Ektoderm zuruckzufuhren. Elektronenmikroskopische Aufnahmen zeigen, das nur das Ektoderm resorbierende Strukturen (Mikrovilli) besitzt. Die Aufnahmeraten waren (von naturlichen Konzentrationen ausgehend) fur die untersuchten Aminosauren verschieden hoch (2–10 μg/g Frischgewicht/h). Die aufgenommene Menge war von der Ausenkonzentration abhangig. Die Konzentrationen, unterhalb denen nur noch eine sehr geringe Aufnahme registriert werden konnte, schwankten fur die einzelnen Aminosauren zwischen 10 und 100 nMol/l. Eine Gruppe von Aminosauren (Phe, Lys, Leu, His, Pro) wurde vorwiegend in den Synthesestoffwechsel eingeschleust, eine andere (Gly und Ser) wurde uberwiegend oxydativen Prozessen zugefuhrt. Der Gehalt an freien Aminosauren von Tentakelgewebe wurde bestimmt. Die Konzentration von Glycin z.B. ist im Gewebe 107mal hoher als im Medium, aus dem noch resorbiert wird. Die Aufnahme erfolgt daher mit groster Wahrscheinlichkeit aktiv. Formale Berechnungen zeigen, das den Tieren durch die Aufnahme geloster organischer Verbindungen ein Energiegewinn erwachst; dieser liegt uber dem Energieverbrauch, der dem Sauerstoffverbrauch aquivalent ist.