The Digestive Diverticula in the Carnivorous Nudibranch, Melibe leonina, Do Not Contain Photosynthetic Symbionts.

A number of nudibranchs, including Melibe engeli and Melibe pilosa, harbor symbiotic photosynthetic zooxanthellae. Melibe leonina spends most of its adult life on seagrass or kelp, capturing planktonic organisms in the water column with a large, tentacle-lined oral hood that brings food to its mouth. M. leonina also has an extensive network of digestive diverticula, located just beneath its translucent integument, that are typically filled with pigmented material likely derived from ingested food. Therefore, the focus of this project was to test the hypothesis that M. leonina accumulates symbiotic photosynthetic dinoflagellates in these diverticula. First, we conducted experiments to determine if M. leonina exhibits a preference for light, which would allow chloroplasts that it might be harboring to carry out photosynthesis. We found that most M. leonina preferred shaded areas and spent less time in direct sunlight. Second, we examined the small green circular structures in cells lining the digestive diverticula. Like chlorophyll, they exhibited autofluorescence when illuminated at 480 nm, and they were also about the same size as chloroplasts and symbiotic zooxanthellae. However, subsequent electron microscopy found no evidence of chloroplasts in the digestive diverticula of M. leonina; the structures exhibiting autofluorescence at 480 nm were most likely heterolysosomes, consistent with normal molluscan digestion. Third, we did not find evidence of altered oxygen consumption or production in M. leonina housed in different light conditions, suggesting the lack of any significant photosynthetic activity in sunlight. Fourth, we examined the contents of the diverticula, using HPLC, thin layer chromatography, and spectroscopy. The results of these studies indicate that the diverticula did not contain any chlorophyll, but rather harbored other pigments, such as astaxanthin, which likely came from crustaceans in their diet. Together, all of these data suggest that M. leonina does sequester pigments from its diet, but not for the purpose of symbiosis with photosynthetic zooxanthellae. Considering the translucent skin of M. leonina, the pigmented diverticula may instead provide camouflage.

Varios nudibranquios, incluidos Melibe engeli y Melibe pilosa, albergan zooxantelas fotosintéticas simbióticas. Melibe leonina pasa la mayoría de su vida adulta en pastos marinos o quelpo, donde captura organismos planctónicos en la columna de agua con una gran capucha oral forrada por tentáculos que llevan comida a su boca. Melibe leonina también tiene una extensa red de divertículos digestivos, ubicados justo debajo de su tegumento translúcido, que generalmente están llenos de material pigmentado probablemente derivado de alimentos ingeridos. Por lo tanto, el objetivo de este proyecto fue evaluar la hipótesis de que M. leonina acumula dinoflagelados fotosintéticos simbióticos en estos divertículos. Primero, realizamos experimentos para determinar si M. leonina se orienta hacia la luz, lo cual permitiría a los cloroplastos que podría albergar el realizar la fotosíntesis. Descubrimos que la mayoría de M. leonina prefería las áreas sombreadas y pasaba menos tiempo bajo la luz solar directa. En segundo lugar, examinamos las pequeñas estructuras circulares verdes en las células que recubren los divertículos digestivos. Al igual que la clorofila, exhibieron autofluorescencia cuando se iluminaban a 480 nm, y también tenían aproximadamente el mismo tamaño que los cloroplastos y las zooxantelas simbióticas. No obstante, la microscopía electrónica no produjo evidencia de cloroplastos en los divertículos digestivos de M. leonina. Es probable que las estructuras que exhibieron autofluorescencia en 480 nm fuesen heterolisosomas, lo cual es consistente con la digestión normal de moluscos. En tercer lugar, no encontramos evidencia de un consumo o producción de oxígeno alterado en M. leonina alojadas varias condiciones lumínicas, lo cual sugiere la ausencia de actividad fotosintética significativa en la presencia de luz solar. En cuarto lugar, examinamos el contenido de los divertículos mediante HPLC, cromatografía en capa fina, y espectroscopia. Los resultados de estos estudios indican que los divertículos no contenían clorofila, pero si otros pigmentos como la astaxantina que probablemente provenía de crustáceos en su dieta. Nuestros datos sugieren que M. leonina secuestra pigmentos de su dieta, pero no con el propósito de la simbiosis con zooxantelas fotosintéticas. Teniendo en cuenta la piel translúcida de M. leonina, los divertículos pigmentados podrían quizás proporcionar camuflaje.

The Distribution and Possible Roles of Small Cardioactive Peptide in the Nudibranch Melibe leonina.

The neuropeptide small cardioactive peptide (SCP) plays an integrative role in exciting various motor programs involved in feeding and locomotion in a number of gastropod species. In this study, immunohistochemistry, using monoclonal antibodies against SCPB, was used to localize SCPB-like-immunoreactive neurons in the central nervous system, and map their connections to various tissues, in the nudibranch, Melibe leonina. Approximately 28-36 SCPB-like-immunoreactive neurons were identified in the M. leonina brain, as well as one large neuron in each of the buccal ganglia. The neuropil of the pedal ganglia contained the most SCPB-like-immunoreactive varicosities, although only a small portion of these were due to SCPB-like-immunoreactive neurons in the same ganglion. This suggests that much of the SCPB-like immunoreactivity in the neuropil of the pedal ganglia was from neurons in other ganglia that projected through the pedal-pedal connectives or the connectives from the cerebral and pleural ganglia. We also observed extensive SCPB innervation along the length of the esophagus. Therefore, we investigated the impact of SCPB on locomotion in intact animals, as well as peristaltic contractions of the isolated esophagus. Injection of intact animals with SCPB at night led to a significant increase in crawling and swimming, compared to control animals injected with saline. Furthermore, perfusion of isolated brains with SCPB initiated expression of the swim motor program. Application of SCPB to the isolated quiescent esophagus initiated rhythmic peristaltic contractions, and this occurred in preparations both with and without the buccal ganglia being attached. All these data, taken together, suggest that SCPB could be released at night to arouse animals and enhance the expression of both feeding and swimming motor programs in M. leonina.

El neuropéptido pequeño péptido cardioactivo (SCP) juega un rol integrativo en inducir varios programas motores involucrados en la alimentación y locomoción de varias especies de gasterópodos. En este estudio se usó inmunohistoquímica con anticuerpos contra SCPB para localizar y describir la conectividad de neuronas con inmunoreacción semejante al SCPB en el sistema nervioso central del nudibranquio Melibe leonina. Identificamos entre 28 y 36 neuronas en el cerebro de M. leonina con inmunoreacción semejantes al SCPB, además de una neurona grande en cada ganglio bucal. El neuropilo del ganglio pedal contiene la mayor cantidad de varicosidades neuronales con inmunoreacción semejante al SCPB aunque sólo una fracción pequeña de éstas provenían de neuronas en el mismo ganglio. Esto sugiere que gran parte de la inmunoreacción semejante al SCPB en el neuropilo del ganglio pedal tienen su origen en neuronas en otros ganglios que enviaron sus proyecciones a través de los conectivos entre los ganglios pedales, o provenientes de los ganglios cerebral o pleural. Observamos además extensa inervación con immunoreacción semejante al SCPB a lo largo del esófago. Por lo tanto investigamos el impacto de SCPB en la locomoción de animales intactos, así también como las contracciones peristálticas en preparaciones de esófagos aislados. Inyecciones nocturnas de SCPB en animales intactos resultó en in incremento significante en rastreo y nado comparado con animales inyectados con soluciones salinas. Más aún, perfusión de cerebros aislados con SCPB inició programas de programas motores de nado. La aplicación de SCPB a preparaciones aisladas de esófagos inactivos inició contracciones peristálticas rítmicas, lo cual ocurrió igualmente en preparaciones con conexiones con el ganglio bucal presentes o ausentes. Tomados en conjunto, nuestros datos sugieren que SCPB podría ser liberado de forma nocturna para despertar los animales y aumentar la expresión de programas motores de alimentación y locomoción en M. leonina.

Detection of salinity by the lobster, Homarus americanus.

Changes in the heart rates of lobsters (Homarus americanus) were used as an indicator that the animals were capable of sensing a reduction in the salinity of the ambient seawater. The typical response to a gradual (1 to 2 ppt/min) reduction in salinity consisted of a rapid increase in heart rate at a mean threshold of 26.6 +/- 0.7 ppt, followed by a reduction in heart rate when the salinity reached 22.1 +/- 0.5 ppt. Animals with lesioned cardioregulatory nerves did not exhibit a cardiac response to changes in salinity. A cardiac response was elicited from lobsters exposed to isotonic chloride-free salines but not to isotonic sodium-, magnesium- or calcium-free salines. There was little change in the blood osmolarity of lobsters when bradycardia occurred, suggesting that the receptors involved are external. Furthermore, lobsters without antennae, antennules, or legs showed typical cardiac responses to low salinity, indicating the receptors are not located in these areas. Lobsters exposed to reductions in the salinity of the ambient seawater while both branchial chambers were perfused with full-strength seawater did not display a cardiac response until the external salinity reached 21.6 +/- 1.8 ppt. In contrast, when their branchial chambers were exposed to reductions in salinity while the external salinity was maintained at normal levels, changes in heart rate were rapidly elicited in response to very small reductions in salinity (down to 29.5 +/- 0.9 ppt in the branchial chamber and 31.5 +/- 0.3 ppt externally). We conclude that the primary receptors responsible for detecting reductions in salinity in H. americanus are located within or near the branchial chambers and are primarily sensitive to chloride ions.

Identifiable nitrergic neurons in the central nervous system of the nudibranch Melibe leonina localized with NADPH-diaphorase histochemistry and nitric oxide synthase immunoreactivity.

Nitric oxide (NO) is a gaseous intercellular messenger produced by the enzyme nitric oxide synthase (NOS). In this study, we used two different techniques-nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and NOS immunocytochemistry-to demonstrate that NOS is present in a pair of identifiable cells in the central nervous system of the nudibranch Melibe leonina. In the Melibe brain, NADPH-d histochemistry revealed only a single pair of bilaterally symmetrical cells in the cerebropleural ganglia. NOS activity also was found in the neuropil of the cerebral, pedal, and buccal ganglia; in the tentacles of the oral hood; in the sensory end of the rhinophores; and in the epithelial tissue of the mouth, preputium, and glans penis. Immunocytochemistry using NOS antisera corroborated the results of the NADPH-d histochemistry by staining the same two cells in the cerebropleural ganglia. Each of these identifiable nitrergic neurons projects into the ipsilateral pedal ganglion. Because the pedal ganglia play a critical role in the control of locomotion, our results provide morphological evidence suggesting that NO may influence swimming or crawling in Melibe leonina.