When morphometry meets taxonomy: morphological variation and species boundaries in Proboscoida (Cnidaria: Hydrozoa)

Species delimitation in marine taxa is often problematic given large intraspecific variation. Based on extensive, recently published genetic sampling from specimens of the hydrozoan families Campanulariidae, Clytiidae and Obeliidae, we evaluate morphological variation in this group, correlating morphometric and phylogenetic patterns for species delimitation. Several species of Campanulariidae are confidently delimited based on differences in size (e.g. Bonneviella species, Tulpa tulipifera and Rhizocaulus verticillatus), while others are re-identified and corroborated based on differences in perisarc thickness (e.g. Silicularia rosea, Orthopyxis and Campanularia species). In Clytiidae, the length and diameter of hydrothecae, height of hydrothecal cusps and perisarc thickness delimit the species Clytia linearis, C. elsaeoswaldae and C. noliformis from others. However, few characters reliably differentiate the clades associated with the nominal species C. gracilis and C. hemisphaerica. In Obeliidae, Obelia geniculata is distinctive in its higher perisarc thickness, and corroborated as a widely distributed species. Obelia longissima and clades refered to O. dichotoma are subtly distinguished, showing a few differences in size and branching of colonies. The taxonomic implications of these results are discussed. With a few exceptions, species can be delimited based on morphometric patterns, once morphological variation is compared.

Comparison of proteomic profiles of the phaeophyte Saccharina japonica thalli proximal to and beneath the front of epiphytic hydrozoan colonies against healthy tissue

The stoloniferous hydrozoan Obelia geniculata commonly colonizes macroalgae such as Saccharina japonica. Each Obelia colony consists of thread-like hydrorhizae attached to the seaweed thallus. The early signaling proteins of epiphytic contamination can be identified using proteomics. To isolate these early signals, parts of the thallus proximal to the hydrozoans were separated from beneath the colony front and from healthy tissue. From the proteomic profiles of S. japonica, we detected 110 protein spots from tissue proximal to hydrozoan colonies (56 increased, 53 decreased, and 1 unchanged in expression relative to healthy tissue) and 133 spots from tissue at the colony front (67 increased, 60 decreased, and 6 unchanged in expression). Of the proteins with increased and decreased expression, SIPA1L1 and actin increased strongly only in the proximal tissues, while NEK2 kinase decreased. CIPK20 and SIPA1L2 increased markedly in both the colony-front and proximal tissues, while CaMK2N2 and GK25369 decreased in both tissues. ATPase β, ADA, kinesin, and HECT domain proteins increased only in the colony-front tissues. Among them, SIPA1L2 increased strongly in both the thallus tissues proximal to the hydrozoans and those beneath the colony front, but was not expressed in response to bryozoan infection.

The permeability to calcium of pigeon erythrocyte ‘ghosts’ studied by using the calcium-activated luminescent protein, obelin

1. Obelin, the Ca2+-activated luminescent protein from the hydroid Obelia geniculata, was sealed inside pigeon erythrocyte ‘ghosts’ in order to investigate effects on their permeability of different methods of preparation and of the bivalent cation ionophore A23187. 2. Changes in free Ca2+ within the ‘ghosts’ were studied by following the rate of luminescence of obelin. The possibility that the obelin might have been released from the ‘ghosts’ during an experiment was investigated by studying the release of inulin and pyruvate kinase from the ‘ghosts’. Less than 10% of the inulin or pyruvate kinase sealed within the ‘ghosts’ was released under any of the experimental conditions. 3. Triton X-100 (0.1–10%, v/v) made the ‘ghosts’ highly permeable to Ca2+. In the presence of 1mm-Ca2+ and Triton, 95–100% of the obelin was utilized within 10–20s. 4. A time-course of resealing ‘ghosts’ at 37°C showed that over a period of 90min, the ‘ghosts’ became gradually less permeable to Ca2+. ‘Ghosts’ which remained at 0°C retained only a small concentration of obelin and ATP, and were highly permeable to Ca2+. 5. Erythrocyte ‘ghosts’ resealed for 30min at 20°C rather than 37°C were more permeable to Ca2+, as shown by the fact that 92% of the obelin in the ‘ghosts’ was utilized during the first 60s after the addition of 1mm-Ca2+, as opposed to 44% for ‘ghosts’ resealed at 37°C. 6. Haemolysis at pH6.0 rather than 7.0 resulted in ‘ghosts’ which were highly permeable to Ca2+ after resealing for 60min at 37°C. Of the obelin in the ‘ghosts’, produced by haemolysis at pH6.0, 90% was utilized in the first 60s after the addition of 1mm-Ca2+ compared with 23% for ‘ghosts’ produced at pH7.0. 7. The bivalent cation ionophore A23187 increased the permeability of the ‘ghosts’ to Ca2+. Maximum effects of the ionophore (16μg/ml) were obtained by preincubating the ‘ghosts’ with the ionophore A23187 (16μg/ml) in the presence of a low concentration of Mg2+ and in the absence of Ca2+.

Extraction, partial purification and properties of obelin, the calcium-activated luminescent protein from the hydroid Obelia geniculata

1. The Ca2+-activated luminescent protein obelin was extracted from the hydroid Obelia geniculata. 2. After the addition of a large excess of calcium (greater than 5mm) a peak in the rate of luminescence occurred within 100ms, followed by an exponential decay (k=2.8s−1). The obelin activity (light emitted) was measured by the peak height or by the total number of counts recorded on a scalar in the first 10s after addition of Ca2+. 3. After an overnight extraction in 40mm-EDTA–200mm-Tris–HCl, pH7.0, 7.2×1011 counts were obtained from 186g of wet hydroids. 4. The stability of the crude extracts was dependent on pH, being optimal at pH7.0. 5. Obelin could be purified threefold with a yield of 69% by selecting the protein precipitated between 60%- and 100%-saturated (NH4)2SO4. The precipitate could be stored for at least 6 months as a suspension in 40mm-EDTA+saturated (NH4)2SO4, pH7.0, frozen at −70°C with a recovery of 95–100%. 6. Luminescence was also stimulated by Sr2+. However, obelin appeared to have a lower affinity for Sr2+ than for Ca2+. Mg2+ inhibited Ca2+-activated luminescence. 7. Obelin could be used to assay as little as 50pmol of Ca2+ in a final volume of 1ml. 8. At pH7.0 in Ca2+–EGTA [ethanedioxybis(ethylamine)tetra-acetate] buffers the rate of obelin luminescence was proportional to the square of the free Ca2+ concentration in the presence and absence of 1 and 10mm-Mg2+. Over the range 0.1–10μm-Ca2+ less than 0.03% of the obelin was consumed/s. 9. In order to use obelin to study free ionized Ca2+ concentrations similar to those found inside cells in the presence of 10mm-Mg2+ a minimum of 108 counts were required. A total of 1012 counts can be readily extracted from about 200g of wet hydroids. Thus a sufficient quantity of an aequorin-like calcium-activated luminescent protein should now be available to workers in the United Kingdom in order to carry out physiological experiments.