Ectonucleotidase Activities Associated with the Olfactory Organ of the Spiny Lobster

The olfactory system of the Florida spiny lobster, Panulirus argus, has olfactory receptors that are excited by the purine nucleotides AMP, ADP, and ATP. These receptors reside on chemosensory neurons that are contained within aesthetasc sensilla on the lateral filaments of the antennules. Also associated with the lobster's olfactory system are ectonucleotidase activities that dephosphorylate excitatory nucleotides, resulting in the production of the nonstimulatory nucleoside adenosine. Our studies of the 5'-ectonucleotidase, ecto-ADPase, and ecto-ATPase activities of this olfactory system showed that each activity was characterized by Michaelis-Menten kinetics; Michaelis constants ranged from 6.9 to 33.5 microM, and maximum velocities ranged from 2.5 to 28.8 fmol/sensillum/s. Evidence that AMP dephosphorylation may serve as an inactivation process was shown by the close correlation between the kinetics of 5'-ectonucleotidase activity and the periodicity of olfactory sampling. Decreased magnesium ion concentration or increased calcium ion concentration resulted in increased ecto-ATPase activity; this activity was insensitive to vanadate ion. Ectonucleotidase activities may have multiple effects on the detection of exogenous nucleotides by a chemosensory system. These effects can be either direct, such as the conversion of an odorant to an inactive compound, or indirect, such as the conversion of an odorant to another compound that can activate or inhibit either receptors or enzymes associated with the system.     

Functional significance of genetically different symbiotic algae Symbiodinium in a coral reef symbiosis

The giant sea anemone Condylactis gigantea associates with members of two clades of the dinoflagellate alga Symbiodinium, either singly or in mixed infection, as revealed by clade-specific quantitative polymerase chain reaction of large subunit ribosomal DNA. To explore the functional significance of this molecular variation, the fate of photosynthetically fixed carbon was investigated by (14)C radiotracer experiments. Symbioses with algae of clades A and B released ca. 30-40% of fixed carbon to the animal tissues. Incorporation into the lipid fraction and the low molecular weight fraction dominated by amino acids was significantly higher in symbioses with algae of clade A than of clade B, suggesting that the genetically different algae in C. gigantea are not functionally equivalent. Symbioses with mixed infections yielded intermediate values, such that this functional trait of the symbiosis can be predicted from the traits of the contributing algae. Coral and sea anemone symbioses with Symbiodinium break down at elevated temperature, a process known as 'coral bleaching'. The functional response of the C. gigantea symbiosis to heat stress varied between the algae of clades A and B, with particularly depressed incorporation of photosynthetic carbon into lipid of the clade B algae, which are more susceptible to high temperature than the algae of clade A. This study provides a first exploration of how the core symbiotic function of photosynthate transfer to the host varies with the genotype of Symbiodinium, an algal symbiont which underpins corals and, hence, coral reef ecosystems.     

The impact of coral bleaching on the pigment profile of the symbiotic alga, Symbiodinium

Bleaching of corals by loss of symbiotic dinoflagellate algae and/or photosynthetic pigments is commonly triggered by elevated temperatures coupled with high irradiance, and is a first-order threat to coral reef communities. In this study, a high-resolution high-performance liquid chromatography method integrated with mass spectrometry was applied to obtain the first definitive identification of chlorophyll and carotenoid pigments of three clades of symbiotic dinoflagellate algae (Symbiodinium) in corals, and their response to experimentally elevated temperature and irradiance. The carotenoids peridinin, dinoxanthin, diadinoxanthin (Dn), diatoxanthin (Dt) and beta-carotene were detected, together with chlorophylls a and c2, and phaeophytin a, in all three algal clades in unstressed corals. On exposure to elevated temperature and irradiance, three coral species (Montastrea franksi and Favia fragum with clade B algae, and Montastrea cavernosa with clade C) bleached by loss of 50-80% of their algal cells, with no significant impact to chlorophyll a or c2, or peridinin in retained algal cells. One species (Agaricia sp. with clade C) showed no significant reduction in algal cells at elevated temperature and irradiance, but lost substantial amounts of chlorophyll a and carotenoid pigments, presumably through photo-oxidative processes. Two coral species (Porites astreoides and Porites porites both bearing clade A algae) did not bleach. The impact of elevated temperature and irradiance on the levels of the photoprotective xanthophylls (Dn + Dt) and beta-carotene varied among the corals, both in pool size and xanthophyll cycling, and was not correlated to coral bleaching resistance.     

Validating Livanow: Molecular Data Agree That Leeches, Branchiobdellidans, and Acanthobdella peledina Form a Monophyletic Group of Oligochaetes

To investigate the phylogenetic relationships of leeches, branchiobdellidans, and acanthobdellidans, whole nuclear 18S rDNA and over 650 bp of mitochondrial cytochrome c oxidase subunit I were acquired from 101 annelids, including 36 leeches, 18 branchiobdellidans, Acanthobdella peledina, as well as 28 oligochaetes and combined with homologous data for 17 polychaete outgroup taxa. Parsimony analysis of the combined aligned dataset supported monophyly of leeches, branchiobdellidans, and acanthobdellidans in 100% of jackknife replicates. Monophyly of the oligochaete order Lumbriculida with Acanthobdellida, Branchiobdellida, and Hirudinea was supported in 84% of jackknife replicates. These results provide support for the hypotheses that leeches and branchiobdellidans are sister groups, that acanthobdellidans are sister to them, and that together with the family Lumbriculidae they all constitute a clade within Oligochaeta. Results support synonymy of the classes Clitellata and the more commonly used Oligochaeta. Leeches branchiobdellidans, and acanthobdellidans should be regarded as orders equal to their closest relatives, the order Lumbriculida.     

Nitric Oxide and Cnidarian-Dinoflagellate Symbioses: Pieces of a Puzzle

The presence of nitric oxide synthase (NOS) activity is demonstratedin the tropical marine cnidarian Aiptasia pallida and in itssymbiotic dinoflagellate algae, Symbiodinium bermudense. Enzymeactivity was assayed by measuring the conversion of arginineto citrulline. Biochemical characterization of NOS from Aiptasiawas characterized with respect to cellular localization, substrateand cofactor requirements, inhibitors, and kinetics. In responseto acute temperature shock, anemones retracted their tentacles.Animals subjected to such stress had lower NOS activities thandid controls. Treatment with NOS inhibitors caused tentacularretraction, while treatment with the NOS substrate L-arginineinhibited this response to stress, as did treatment with NOdonors. These results provide a preliminary biochemical characterizationof, and suggest a functional significance for, NOS activityin anthozoan-algal symbiotic assemblages.     

Molecular quantification of symbiotic dinoflagellate algae of the genus Symbiodinium

The dinoflagellate microalga Symbiodinium is the dominant algal symbiont in corals and related marine animals. To explore the incidence of mixed infections, methods employing real-time quantitative polymerase chain reaction (QPCR) and fluorescence in situ hybridization (FISH) were developed. In experiments focusing on Symbiodinium clades A and B, QPCR and FISH results were well correlated and generally more precise and sensitive than those from the endpoint PCR-restriction fragment length polymorphism analysis (PCR-RFLP) traditionally used for this application, thus increasing the detected incidence of mixed infections. For example, the prevalence of mixed infections in the sea anemone Condylactis gigantea was 40% by PCR-RFLP and 80%-90% by QPCR and FISH. However, the use of QPCR and FISH was limited by inter-host variation in the rRNA gene copy number per Symbiodinium cell, precluding any single conversion factor between QPCR signal and Symbiodinium cell number; and one FISH probe that gave excellent hybridization efficiency with cultured Symbiodinium yielded variable results with Symbiodinium from symbioses. After controlling for these caveats, QPCR studies revealed that field-collected hosts previously described as universally unialgal bore up to 1.6% of the alternative clade. Further research is required to establish the contribution that algal cells at low density in symbiosis and external to the symbiosis make to the minor clade.     

Biochemistry of an Olfactory Purinergic System: Dephosphorylation of Excitatory Nucleotides and Uptake of Adenosine

Abstract: The olfactory organ of the spiny lobster, Panu-lirus argus , is composed of chemosensory sensilla containing the dendrites of primary chemosensory neurons. Receptors on these dendrites are activated by the nucleotides AMP, ADP, and ATP but not by the nucleoside adenosine. It is shown here that the lobster chemosensory sensilla contain enzymes that dephosphorylate excitatory nucleotides and an uptake system that internalizes the nonexcitatory dephosphorylated product adenosine. The uptake of [³H]-adenosine is saturable with increasing concentration, linear with time for up to 3h, sodium dependent, insensitive to moderate pH changes and has a K _m of 7.1 μ M and a V_max of 5.2 fmol/sensillum/min (573 fmol/μg of protein/min). Double-label experiments show that sensilla dephosphorylate nucleotides extracellularly; ³H from adenine-labeled AMP or ATP is internalized, whereas ³²P from phosphate-labeled nucleotides is not. The dephosphorylation of AMP is very rapid; ³H from AMP is internalized at the same rate as ³H from adenosine. Sensillar 5'-ectonucleotidase activity is inhibited by ADP and the ADP analog α,β-methylene ADP. Collectively, these results indicate that the enizymes and the uptake system whereby chemosensory sensilla of the lobster inactivate excitatory nucleotides and clear adenosine from extracellular spaces are very similar to those present in the internal tissues of vertebrates, where nucleotides have many neuroactive effects.     

Validating Livanow: molecular data agree that leeches, Branchiobdellidans, and Acanthobdella peledina form a monophyletic group of oligochaetes.

To investigate the phylogenetic relationships of leeches, branchiobdellidans, and acanthobdellidans, whole nuclear 18S rDNA and over 650 bp of mitochondrial cytochrome c oxidase subunit I were acquired from 101 annelids, including 36 leeches, 18 branchiobdellidans, Acanthobdella peledina, as well as 28 oligochaetes and combined with homologous data for 17 polychaete outgroup taxa. Parsimony analysis of the combined aligned dataset supported monophyly of leeches, branchiobdellidans, and acanthobdellidans in 100% of jackknife replicates. Monophyly of the oligochaete order Lumbriculida with Acanthobdellida, Branchiobdellida, and Hirudinea was supported in 84% of jackknife replicates. These results provide support for the hypotheses that leeches and branchiobdellidans are sister groups, that acanthobdellidans are sister to them, and that together with the family Lumbriculidae they all constitute a clade within Oligochaeta. Results support synonymy of the classes Clitellata and the more commonly used Oligochaeta. Leeches branchiobdellidans, and acanthobdellidans should be regarded as orders equal to their closest relatives, the order Lumbriculida.     

Morphological characteristics facilitating stimulus access and removal in the olfactory organ of the spiny lobster, Panulirus argus: insight from the design

The olfactory organ (antennule) of the spiny lobster, Panulirusargus, has from 1000–2000 olfactory sensilla (aesthetascs)which are grouped in a dense tuft along the distal portion ofthe lateral filament. This assemblage of aesthetascs, togetherwith other associated sensilla, forms a substantial boundarylayer through which odor stimuli must diffuse in moving to andfrom the aesthetascs. Periodic flicking of the antennule, abehavior analogous to sniffing in certain vertebrate species,is considered to be a means of reducing the thickness of thisboundary layer. In this report we describe the structure ofthe aesthetasc tuft and examine certain of its dynamic properties.We propose that the unique configuration of the aesthetasces,together with their orientation, serves to channel water flowbetween these sensilla during a flick, thereby reducing diffusiondistances and consequently facilitating the access and removalof odor stimuli in a rapid, synchronized manner. The functionalsignificance of this and other design features of the aesthetasctuft is considered in light of the current understanding offundamental olfactory process.