Chitobiase activity in the epidermis and hepatopancreas of the fiddler crab Uca pugilator during the molting cycle

The activity of chitobiase, also known as N-acetyl-β-glucosaminidase, in the epidermis and hepatopancreas of the fiddler crab Uca pugilator (Bosc, 1802), during the molting cycle, was investigated. A pH optimum of 5 to 6 was found for the enzymatic activity in both the epidermis and hepatopancreas. The temperature optimum for epidermal and hepatopancreatic chitobiase activities was 50 to 60 °C. The K _m values for epidermal and hepatopancreatic chitobiase activities at 19 °C were 0.190 ± 0.027 and 0.203 ± 0.016 mM 4-methylumbelliferyl-N-acetyl-β-glucosaminide, respectively. Hepatopancreatic chitobiase activity was significantly higher than epidermal enzymatic activity in all the molt cycle stages tested except Postmolt Stage A-B. Chitobiase activity varied significantly during the molting cycle, with the epidermal enzymatic activity in Premolt Stage D_3–4 significantly higher than in Stage C (intermolt) and Premolt Stage D₀, whereas hepatopancreatic chitobiase activity in Premolt Stage D_3–4 was significantly higher than in all other molt stages tested. The patterns of chitobiase activity in the epidermis and hepatopancreas correlate well with the hemolymph titer of ecdysteroids in U. pugilator during the molting cycle; this suggests that chitobiase activity in both tissues is regulated at least in part by the steroid molting hormones. Received: 6 May 1998 / Accepted: 12 September 1998     

Heat production in Saduria entomon (Isopoda) from the Gulf of Gdańsk during an experimental exposure to anoxic conditions

The benthic crustacean Saduria entomon occurs frequently in deeper parts of the Gulf of Gdańsk. It is one of a few species able to survive oxygen deficiencies in its natural environment. The anaerobic heat production of S. entomon during 40 h of anoxia was determined. Additionally, the effects of size, sex and activity of the organism on its heat production were investigated. Average heat production of S. entomon was 0.25 ± 0.16 mJ g⁻¹ wet wt s⁻¹ (n=55, avg. length 39 ± 6 mm). The amount of heat produced decreased with increasing body size. The heat production of S. entomon males was greater than that of females ( p<0.05). Specimens kept in chambers with glass beads and water were less active and had lower metabolic rates than those placed in chambers containing water only (more active). During 40 h of anoxia S. entomon gradually decreased its heat production to 5–16% of aerobic level, demonstrating the high adaptation of this species to changeable oxygen conditions in the Baltic Sea. Received: 31 July 1997 / Accepted: 21 January 1998     

Interactions between NH+4 and NO−3 uptake and assimilation: comparison of diatoms and dinoflagellates at several growth temperatures

Ammonium concentrations of ∼1 M are commonly cited as being the threshold for inhibition of NO₃ ⁻ uptake, but the applicability of this threshold to phytoplankton from different taxonomic classes has rarely been examined. Additionally, little is known about the influence of environmental variables (e.g. growth temperature) on the interaction between ambient NH₄ ⁺ and NO₃ ⁻ uptake. Four species of estuarine phytoplankton, two diatom [Chaetoceros sp., and Thalassiosira weissflogii (Grunow) Fryxell et Hasle] and two dinoflagellate [Prorocentrum minimum (Pavillard) Schiller, and Gyrodinium uncatenum Hulburt], were grown on NO₃ ⁻ at several different temperatures (4, 10, 15, or 20 °C), and the impact of NH₄ ⁺ additions on NO₃ ⁻ uptake/assimilation (non-TCA-extracted) and assimilation (TCA-extracted) was assessed. For all species at all temperatures, NO₃ ⁻ uptake/assimilation and assimilation rates decreased in a roughly exponential manner with increasing NH₄ ⁺ concentrations but were not completely inhibited even at elevated NH₄ ⁺ concentrations of 200 μM. Estimated half-inhibition concentrations (K _i) were significantly greater in the diatom species (mean ± SE; 2.70 ± 0.67 μM) than in the dinoflagellate species (1.26 ± 0.55 μM). Half-inhibition constants were positively related to temperature-limited relative growth rate although not significantly. The observed inhibition of NO₃ ⁻ uptake and assimilation, as a percentage of NO₃ ⁻ uptake in the absence of NH₄ ⁺, averaged about 80% and ranged from 49 to 100%. For all species, a significant (P < 0.001) positive correlation was found between percent inhibition of NO₃ ⁻ assimilation and temperature-limited relative growth rate. Two experiments on Chesapeake Bay phytoplankton during an April 1998 diatom bloom showed that in short-term (∼1 h) temperature manipulation experiments, percent inhibition of NO₃ ⁻ uptake/assimilation was also positively related (P = 0.05) to experimental temperature. The observed relationships between temperature-limited relative growth rate and percent inhibition of NO₃ ⁻ assimilation rates for the species tested suggest that at the enzyme level, the inhibitory mechanism of NO₃ ⁻ assimilation is similar among species, but at the whole cell level may be regulated by species-specific differences in the accumulation of internal metabolites. These findings add not only to our understanding of species-specific variability and the role of growth temperature, but also provide additional data with which to evaluate current models of NH₄ ⁺ and NO₃ ⁻ interactions. Received: 31 August 1998 / Accepted: 7 December 1998     

Field distribution and sulphide tolerance of Capitella capitata (Annelida: Polychaeta) around shallow water hydrothermal vents off Milos (Aegean Sea). A new sibling species?

The cosmopolitan polychaete Capitella capitata, known as a complex of opportunistic sibling species, usually dominates the macrobenthos of polluted or unpredictable environments. A population of C. capitata, termed Capitella sp. M, was found in a shallow water hydrothermal vent area south of Milos (Greece). Here, this population occurs close to vent outlets (termed the “transition zone”), an environment with steep gradients of temperature, salinity and pH and increased sulphide concentrations of up to 710 μM. The field distribution of C. capitata in relation to sulphide concentrations around the vent outlets was investigated and sulphide tolerance experiments were conducted on laboratory-cultured worms to elucidate possible adaptations of Capitella sp. M to these extreme environmental conditions. In order to investigate whether the population from the Milos hydrothermal vent area can be considered a distinct sibling species within the C. capitata complex, crossbreeding experiments and analysis of general protein patterns were conducted with Capitella sp. M and three other C. capitata populations of different ecological ranges. Capitella sp. M showed high resistance (median survival time: 107 ± 38 h) to anoxia plus high sulphide concentrations of 740 μM. It seems that the ability to survive high-sulphide conditions in combination with reduced interspecific competition enables the polychaete to maintain a continuous population in this rigorous habitat. From the extremely high tolerance to anoxia and sulphide, shown in both the crossbreeding experiments and the analysis of total proteins, it can be concluded that Capitella sp. M from the Milos hydrothermal vent area represents a separate sibling species within the C. capitata complex. Received: 3 March 1997 / Accepted: 12 September 1997     

Influence of cadmium accumulation and dietary status on fatty acid composition in two colour forms of shore crabs, Carcinus maenas

 The effects of cadmium exposure and dietary status on cadmium accumulation, fatty acid (FA) content and profiles were investigated in two colour forms of the shore crab Carcinus maenas. Groups of shore crabs were either starved or fed with blue mussels, Mytilus edulis, during a 40 d exposure period to 2 or 6 μM Cd²⁺ (as CdCl₂). Starved green individuals accumulated more cadmium in haemolymph and hepatopancreas than did red crabs and green crabs fed during the experiments. In the red colour form, no difference in cadmium accumulation was observed between starved and fed individuals. In both colour forms, hepatopancreas contained more FA than gills and muscle. The FAs often present in the largest amounts in the tissues were 16:0, 16:1ω7, 18:1ω7, 18:1ω9, 20:4ω6, 20:5ω3 and 22:6ω3. However, saturated (SAFA) and mono-unsaturated fatty acids (MUFAs) were dominant in hepatopancreas, whereas poly-unsaturated fatty acids (PUFAs) were dominant in gills and muscles. At the beginning of the experiment, the total FA content in the hepatopancreas was 111.6 mg g⁻¹ (dry weight) for red crabs and 78.4 mg g⁻¹ for green shore crabs. During the experiment, however, the FA content decreased in red crabs. This decrease was more pronounced for starved individuals than for fed individuals. Also, the decrease in FA content was more pronounced in crabs exposed to 6 μM cadmium compared to crabs exposed to 2 μM or crabs not exposed to cadmium. No change in FA content was observed in green shore crabs, irrespective of diet and cadmium exposure. For both colour forms, no change in FA content was observed for gills and muscle. In red crabs, a decrease was observed for all FAs in the hepatopancreas. This decrease, however, was more pronounced for SAFAs and MUFAs than for PUFAs, indicating that the metabolism of FAs during starvation and cadmium exposure is selective. The experiments indicate that green colour forms of shore crabs are more tolerant of natural stress such as starvation and anthropogenic stress, e.g. cadmium exposure, than are red colour forms of shore crabs. Received: 23 September 1999 / Accepted: 29 April 2000     

Purification and partial characterisation of phenoloxidase from the colonial ascidian Botryllus schlosseri

Phenoloxidase (PO) from the colonial ascidian Botryllus schlosseri was purified using two different chromatographic strategies. A three-step purification was developed in order to maintain enzyme activity, whereas an easier purification procedure was adopted to obtain enough PO for the production of specific polyclonal antibodies. The enzyme showed optimal pH and temperature values of 7.0 to 7.5 and 35 °C, respectively, and a K _m value of 4.62 ± 0.76 mM was estimated using l-DOPA as substrate. A molecular weight of 160 kDa was determined after SDS-PAGE under non-reducing conditions. The addition of the reducing agent β-mercaptoethanol caused the disappearance of the 160 kDa band and the appearance of a new band at 80 kDa, suggesting that active PO is a dimer and the two subunits are linked by disulphide bridges. Received: 14 December 1998 / Accepted: 24 August 1999     

Low molecular-weight factor from Plesiastrea versipora (Scleractinia) that modifies release and glycerol metabolism of isolated symbiotic algae

When symbiotic dinoflagellate algae (Symbiodinium sp., isolated from the coral Plesiastrea versipora) were incubated with NaH¹⁴CO₃ in the light in seawater, they released 22.69±9.16 nmol carbon/10⁶ algae. Release of photosynthetically fixed carbon was stimulated more than six-fold for algae incubated in host-tissue homogenate (148.54±97.03 nmol C/10⁶ algae) and more than four-fold (102.00±49.16 nmol C/10⁶ algae) for algae incubated in a low molecular weight fraction (≤1 000 M _r ) prepared from host homogenate. Soluble released ¹⁴C-labelled products, as determined by chromatography and autoradiography, were the same when algae were incubated in either host homogenate or the low molecular weight fraction. After 4 h incubation in the light (300 mol photons m⁻² s⁻¹),␣intracellular␣glycerol increased in algae incubated with the low molecular weight fraction (an increase of 0.39 to␣0.67 nmol glycerol/10⁶ algae) compared with little or no increase in algae incubated in seawater (0 to 0.12 nmol glycerol/10⁶ algae). Partial inhibition of triglyceride synthesis (up to 51%) was also observed when algae were incubated in the low molecular weight fraction. All these effects are the same as those observed when algae were incubated in host homogenate. These data indicate that the “host release-factor” activity of P.␣versipora is a compound of low molecular weight. Received: 13 February 1997 / Accepted: 24 October 1997     

Survival of the intertidal pulmonate Onchidium tumidium during short term and long term anoxic stress

Onchidium tumidium showed a triphasic response to anoxia. Twelve hours of anoxic exposure had no effect on the glycogen content in O. tumidium. However, there were significant increases in the alanine, lactate and succinate contents in the anoxic individuals. These were accompanied by a significant decrease in the ATP content. These results suggest that O. tumidium survived the first 12 h of anoxic exposure without increasing the glycolytic flux to compensate for the lower efficiency of ATP production through anaerobic pathways. Indeed, the fructose-2,6-bisphosphate (F-2,6-P₂) content and the percentage of phosphofruc-tokinase (PFK) associated with the subcellular particles remain unchanged in O. tumidium exposed to 12 h of anoxia. Hence, a reduction in the metabolic rate of these individuals might have occurred during such a period of anoxia. In contrast, in between 12 and 24 h of anoxic exposure, the glycogen content O. tumidium decreased significantly, and levelled off thereafter. A significant increase in the percentage of PFK associated with the subcellular particles was observed in individuals exposed to 24 h of anoxia. In addition, the F-2,6-P₂ content of these anoxic individuals increased significantly. Taken together, these two mechanisms could activate PFK and lead to a greater glycolytic flux. Beyond 24 h of anoxic exposure, survival of O. tumidium must have required considerable suppression of metabolism as accumulation of end products and depletions of glycogen and ATP had reached constant levels.     

Effect of Ca2+ on survival and development of metamorphosing bonefish (Albula sp.) leptocephali

Bonefish (Albula sp.) larvae (leptocephali) from the Gulf of California complete metamorphosis in ˜10 d in natural seawater (35‰S; Ca²⁺ conc = 10.5 mM). The increase in ossification that occurs near the end of the non-feeding metamorphic period, in addition to the ability of larvae to complete metamorphosis in dilute seawater (8‰ S) prompted the present study, where the effects of varying the external calcium ion concentration, [Ca²⁺]_e, of artificial seawater (ASW) on the survival, development and internal (whole-body) calcium ion content, (Ca²⁺)_i, of unfed metamorphosing larvae were investigated. Early-metamorphosing larvae placed in␣ASW, where [Ca²⁺]_e = 10.1 mM, survived for up to 10 d and developed normally without exogenous nutrients. In shorter-term experiments (4 to 5 d), no differences in survival were found for larvae in ASW with [Ca²⁺]_e rang-ing from 1.5 to 10.1 mM. However, in Ca²⁺-free ASW, most larvae died within 27 h and no larvae survived more than 42 h; the median lethal time (LT₅₀), and its 95% confidence limits, were 14.5 (10.0 to 20.9) h. High mortality (81% after 20 h) also occurred in 1.0 mM Ca²⁺ ASW, but 2 of 16 larvae tested survived for 96 h. The 96 h median tolerance limit (TL_M), corrected for control mortality, was 1.2 mM Ca²⁺. In natural seawater, larval (Ca²⁺)_i remained relatively constant ( = 0.419 mg larva⁻¹)␣in early- and intermediate-metamorphosing larvae, and then increased to a mean value of 0.739 mg larva⁻¹ in advanced larvae, indicating that Ca²⁺ was␣taken up from the medium at this stage; the increase in (Ca²⁺)_i corresponded to the period of ossification of the vertebral column. Internal (whole-body) magnesium ion content (Mg²⁺)_i showed no significant change during metamorphosis ( = 0.089 mg larva⁻¹). No significant differences in (Ca²⁺)_i were found in advanced larvae in natural seawater and those in ASW, with [Ca²⁺]_e ranging from 2.0 to 10.1 mM. However, clearing and staining revealed that ossification of the vertebral column had not yet occurred in advanced larvae from 2.0 to 10.1 mM Ca²⁺ ASW. Also, low [Ca²⁺]_e (1.0 to 2.0 mM) usually produced deformed larvae that swam erratically, at times showing “whirling” behavior. Received: 21 August 1996 / Accepted: 26 August 1996     

Sociality,Bateman’s gradients,and the polygynandrous genetic mating system of round-tailed ground squirrels (<Emphasis Type="Italic">Xerospermophilus tereticaudus</Emphasis>)

Historically, most mammals have been classified as polygynous; although recent molecular evidence suggests that many mammals may be polygynandrous, particularly the ground-dwelling sciurids. We genotyped 351 round-tailed ground squirrels (Xerospermophilus tereticaudus) using seven microsatellite loci to determine paternity in 31 litters from 2004 to 2007. Polygyny was evident in all years except in 2007, when the population size was reduced. Multiple paternity occurred in the majority of litters (55%) with 2.5 ± 0.26 sires/litter (n = 31). Forty-nine percent of resident males (n = 114) sired offspring, and of males that sired offspring (n = 56) 27% sired young in multiple litters in a single breeding season. Litter size was positively correlated with the number of sires. Through an indirect analysis of paternity, we found 21 litters (68%) with an average relatedness of 0.5 or less. Males had a greater opportunity for sexual selection (I _s = 1.60) than females (I _s = 0.40); Bateman’s gradient was also greater in males (1.07 ± 0.04, n = 56) than females (0.82 ± 0.08, n = 31). The mating system in round-tailed ground squirrels defined through genetic analyses and Bateman’s gradients is polygynandrous compared to the previously suggested polygynous mating system as established by behavioral observations and fits within the predictions of the ground squirrel sociality models. Upon evaluating the predictions of the sociality models among sciurid species, we found a negative relationship between the level of sociality with litter size and the average percentage of multiple paternity within a litter. Thus, recent genetic information and reclassification of mating systems support the predictions of the ground-dwelling squirrel sociality models.     

Role of protein in larval swimming and metamorphosis of Bugula neritina (Bryozoa: Cheilostomatida)

Larvae of the marine cheilostomatid bryozoan Bugula neritina (L.) were prevented from settling for 1, 4 and 8 h by mechanical agitation, following which settlement and metamorphosis success were examined. Settlement rates were significantly affected by swimming time, which decreased from 100% after 2 h to 93.7 ± 4.3% after 8 h. Similarly, metamorphosis to the feeding ancestrula was significantly impaired following a swimming time of 8 h, declining from 93.7 ± 4.3% after 1 h to 65.9 ± 7.0% after 8 h. The resultant colonies grew well for the first 3 wk, following which time, growth patterns became erratic. Growth rate was in all cases highly variable, and did not correlate with enforced swimming times. Larval protein composition was examined after 1, 4 and 8 h swimming time, and post-larval composition 1, 2, 5, 24 and 48 h after settlement using sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis (SDS-PAGE). Individual protein content was measured using a densitometer. Larvae did not consume protein during swimming, however a protein measuring 170 kdaltons was consumed during metamorphosis. These results are discussed in the context of larval settlement and energetics. Received: 19 July 1998 / Accepted: 3 December 1998     

Selective feeding in shellfish: size-dependent rejection of large particles within pseudofaeces from Mytilus edulis, Ruditapes philippinarum and Tapes decussatus

Mytilus edulis L., Ruditapes philippinarum (Adams & Reeve) and Tapes decussatus L. were fed particles of the same shape (spherical), the same density (2.1 g cm⁻³) and the same chemical composition (SiO₂), but which varied in diameter from 5 to 37 μm. Findings obtained at different particle concentrations (mean ± SD) of 51 ± 2, 105 ± 18 and 171 ± 17 mg l⁻¹ invariably indicate that significant proportions of all particles with diameters larger than from between 7.5 and 22.5 μm were preferentially rejected as pseudofaeces. We define the preferential ingestion index (PII) as the ratio between average particle volume in pseudofaeces and average particle volume in food. Whatever the particle concentration or the species, this PII was always statistically higher than 1. Irrespective of particle concentration, PII values in M. edulis were lower than in T. decussatus (averages of 1.2 and 2, respectively). PII values in M. edulis were also lower than in R. philippinarum maintained at particle concentrations above 171 ± 17 mg l⁻¹. We suggest that preferential size-dependent rejection of larger particles could be of significant adaptive value in the natural environment, either if there are large inorganic particles, or if the average organic content of smaller particles is higher. Received: 11 January 1997 / Accepted: 8 March 1997     

Mitochondrial DNA analysis of the genetic relationships among populations of scad mackerel (Decapterus macarellus, D. macrosoma, and D. russelli) in South-East Asia

The genetic relationships among South-East Asian populations of the scad mackerels Decapterus macarellus, D. macrosoma and D. russelli (Pisces: Carangidae) were investigated. In 1995 and 1996, 216 fish were sampled in seven localities spanning the seas of Indonesia and were examined for restriction-site polymorphisms using ten restriction enzymes for the mitochondrial (mt) DNA control region, amplified by the polymerase chain-reaction. The inferred phylogeny of haplotypes led to the recognition of three distinct mitochondrial lineages or phylads consistent with the distinctions of current taxonomy. All 15 mtDNA haplotypes found in D. macarellus and all 9 haplotypes found in D. macrosoma were arranged as star-like clusters, suggesting recent evolutionary history. In contrast, the phylad formed by 6 haplotypes in D. russelli from the Sulawesi Sea exhibited diffuse topology, suggesting that ancestral lineages of this species have been retained to the present. Average nucleotide-divergence estimates between haplotypes of different phylads were between 0.042 and 0.135, suggesting ancient separation, in consistency with published allozyme data. High levels of haplotype diversity, but no geographical heterogeneity, was detected within D. macarellus from the Molucca Sea and the Banda Sea. Populations of D. macrosoma exhibited both significant differences between adjacent regions (Sunda Strait and Java Sea), and broadscale genetic homogeneity from the South China Sea to the Sulawesi Sea via the Java Sea and Makassar Strait. The geographic isolation of the D. macrosoma population sampled in the Sunda Strait suggests that this region constitute a sharp transition zone between the Indian Ocean and the Sunda Shelf. Near-monomorphism of haplotypes and low nucleotide diversity (d _X) were observed in the samples of D. macrosoma from the continental shelf (haplotype-diversity estimates, h, = 0.00 to 0.25 ± 0.08 and d _X = 0.000 to 0.002). This was in contrast to the comparatively high haplotype and nucleotide diversities observed in other pelagic fish species including D. macarellus (h = 0.82 ± 0.05, d _X = 0.012 to 0.015) and D. russelli (h = 0.63 ± 0.12, d _X = 0.016), and in the oceanic D. macrosoma population sampled in the Sunda Strait (h = 0.67 ± 0.31, d _X = 0.005). We hypothesise that this may be the consequence of recent and perhaps repeated bottleneck events that have affected the D. macrosoma population sampled on the continental shelf. Received: 29 September 1997 / Accepted: 3 September 1999     

Microsensor studies of photosynthesis and respiration in the symbiotic foraminifer Orbulina universa

Oxygen and pH microelectrodes were used to investigate the microenvironment of the planktonic foraminifer Orbulina universa and its dinoflagellate endosymbionts. A diffusive boundary layer surrounds the foraminiferal shell and limits the O₂ and proton transport from the shell to the ambient seawater and vice versa. Due to symbiont photosynthesis, high O₂ concentrations of up to 206% air saturation and a pH of up to 8.8, i.e. 0.5 pH units above ambient seawater, were measured at the shell surface of the foraminifer at saturating irradiances. The respiration of the host–symbiont system in darkness decreased the O₂ concentration at the shell surface to <70% of the oxygen content in the surrounding air-saturated water. The pH at the shell surface dropped to 7.9 in darkness. We measured a mean gross photosynthetic rate of 8.5 ± 4.0 nmol O₂ h⁻¹ foraminifer⁻¹. The net photosynthesis averaged 5.3 ± 2.7 nmol O₂ h⁻¹. In the light, the calculated respiration rates reached 3.9 ± 1.9 nmol O₂h⁻¹, whereas the dark respiration rates were significantly lower (1.7 ± 0.7 nmol O₂ h⁻¹). Experimental light–dark cycles demonstrated a very dynamic response of the symbionts to changing light conditions. Gross photosynthesis versus scalar irradiance curves (P vs E _o curves) showed light saturation irradiances (E _k) of 75 and 137 μmol photons m⁻² s⁻¹ in two O. universa specimens, respectively. No inhibition of photosynthesis was observed at irradiance levels up to 700 μmol photons m⁻² s⁻¹. The light compensation point of the symbiotic association was 50 μmol photons m⁻² s⁻¹. Radial profile measurements of scalar irradiance (E _o) inside the foraminifera showed a slight increase at the shell surface up to 105% of the incident irradiance (E _d). Received: 26 January 1998 / Accepted: 11 April 1998     

Cell cycle and toxin production in the benthic dinoflagellate Prorocentrum lima

Profiles of diarrhetic shellfish poisoning (DSP) toxins produced throughout the growth cycle and the cell cycle of the toxigenic marine dinoflagellate Prorocentrum lima were studied in triplicate unialgal batch cultures. Cells were pre-conditioned at 18 ± 1 °C, under a photon flux density (PFD) of 90 ± 5 μmol m⁻² s⁻¹ on a 14 h light:10 h dark photoperiod. In exponential growth phase, cultures were synchronized in darkness for 17 d. After dark synchronization, cultures were transferred back to the original photoperiod regime. Cells were harvested for DSP toxin analysis by LC-MS (liquid chromatography with mass spectrometry), and double-stranded (nuclear) DNA was quantified by flow cytometry. The cell populations became asynchronous within approximately 3 d after transition from darkness to the 14 h light:10 h dark photoperiod. This may be due to the prolonged division cycle (5 to 7 d) that is not tightly phased by the photoperiod. Unlike other planktonic Prorocentrum spp., cytokinesis in P. lima occurred early in the dark and ceased by “midnight”. Cellular levels of the four principal DSP toxins, okadaic acid (OA), OA C8-diol-ester (OA-D8), dinophysistoxin-1 (DTX1) and dinophysistoxin-4 (DTX4), ranged from 0.37 to 6.6, 0.02 to 1.5, 0.04 to 2.6, and 1.8 to 7.8 fmol cell⁻¹, respectively. No toxin production was evident during the extended period of dark synchronization nor during the initial period when NH₄ was consumed as the major nitrogen source. Soon after the cells were returned to the 14 h light:10 h dark cycle and they began to take up NO₃, cellular levels of all four toxins gradually increased. This increase in DSP toxins usually occurred in the light, marked by a rise in DTX4 levels that preceded an increase in the cellular concentration of OA and DTX1 (delayed by 3 to 6 h). Thus, DTX4 synthesis is initiated in the G1 phase of the cell cycle and persists into S phase (“morning” of the photoperiod), whereas OA and DTX1 production occurs later during S and G2 phases (“afternoon”). No toxin production was measured during cytokinesis, which happened early in the dark. The evidence indicates that toxin synthesis is restricted to the light period and is coupled to cell cycle events. Received: 3 September 1998 / Accepted: 30 March 1999     

Timing of metamorphosis and larval segregation of the Atlantic eels Anguilla rostrata and A. anguilla, as revealed by otolith microstructure and microchemistry

Otolith microstructure and microchemistry were examined in juveniles of American (Anguilla rostrata) and European (A. anguilla) eels. Otolith increment width markedly increased from age 132 to 191 d (156 ± 18.9 d; mean ± SD) in A. rostrata and 163 to 235 d (198 ± 27.4 d; mean ± SD) in A. anguilla, both of which were coincident with drastic decreases in otolith Sr:Ca ratios, suggesting that metamorphosis from leptocephalus to glass eel began at those ages in each species. The duration of metamorphosis was estimated to be 18 to 52 d from otolith microstructure, for both species studied. Ages at recruitment were 171 to 252 d (206 ± 22.3 d; mean ± SD) in A. rostrata and 220 to 281 d (249 ± 22.6 d; mean ± SD) in A. anguilla. In these two species, positive linear relationships were found in ages between the beginning of metamorphosis and recruitment, suggesting that early metamorphosing larvae recruited at younger ages. Duration of the leptocephalus stage to recruitment in A. anguilla was about 40 d longer than that in A. rostrata. The geographical segregation between the two species in the Atlantic Ocean seems to be involved in the differences in the duration of the leptocephalus stage (age at metamorphosis). Received: 8 November 1999 / Accepted: 8 May 2000     

Effects of dissolved ammonium addition and host feeding with Artemia salina on photoacclimation of the hermatypic coral Stylophora pistillata

 Effects of nutrient treatments on photoacclimation of the hermatypic coral Stylophora pistillata (Esper) were studied. Studies on photoacclimation of colonies from different light regimes in the field were evaluated and used to design laboratory experiments. Coral colonies were collected in the Gulf of Eilat (Israel) from January to March 1993. Exterior branches of colonies from different depths (1 to 40 m) displayed different trends in production characteristics at reduced and very low levels of illumination. From 24 ± 3% to 12 ± 2% of incident surface photosynthetic active radiation (PAR_o), zooxanthella population density and chlorophyll a+c per 10⁶ zooxanthellae increased, a trend seen in the range of light levels optimal for coral growth (90 to 30% PAR_o). The P _max of CO₂ per 10⁶ zooxanthellae decreased, while P _max of CO₂ per 10³ polyps increased, indicating an increase in zooxanthella population density at low light levels. Proliferous zooxanthella frequency (PZF, a measure of zooxanthella division) declined significantly at light levels <18 ± 3% PAR_o. At the lowest levels of illumination (<5% PAR_o), zooxanthella population density decreased, as did the PZF; chl a+c per 10⁶ zooxanthellae was unchanged. In 28-d experiments, exterior coral branches from the upper surfaces of colonies from 3 m depth (65 ± 4% PAR_o) were incubated in aquaria under bright (80 to 90% PAR_o), reduced (20 to 30% PAR_o), and extremely low (2 to 4% PAR_o) light intensities. At each light intensity, the corals were maintained in three feeding treatments: sea water (SW); ammonium enriched SW (SW + N); SW with Artemia salina nauplii (SW + A). An increase in P _max of CO₂ per 10³ polyps was found in corals acclimated to reduced light (20 to 30% PAR_o) in nutrient-enriched SW, while in SW, where the increase in zooxanthella population density was smaller, it did not occur. Nutrient enrichments (SW + N at 2 to 4% PAR_o and SW + A at 20 to 30% PAR_o) increased zooxanthella population density, but had no effect on chl a+c per 10⁶ zooxanthellae. Acclimation for 14 d to reduced (10 to 20% PAR_o) and extremely low (1 to 3% PAR_o) light intensities shifted ¹⁴C photoassimilation into glycerol and other compounds (probably glycerides), rather than sugars. Both ammonium addition and feeding with Artemia salina nauplii resulted in an increase in photosynthetic assimilation of ¹⁴C into amino acids. We conclude that acclimation to reduced light consists of two processes: an increase in photosynthetic pigments and in zooxanthella population density. Both processes require nitrogen, the increase in zooxanthella population density needing more; this adaptation is therefore limited in nitrogen-poor sea water. Received: 19 June 1998 / Accepted: 13 June 2000     

Carbon dioxide use by chemoautotrophic endosymbionts of hydrothermal vent vestimentiferans: affinities for carbon dioxide, absence of carboxysomes, and δ13C values

The hydrothermal vent vestimentiferans Riftia pachyptila Jones, 1981 and Ridgeia piscesae Jones, 1985 live in habitats with different abundances of external CO₂. R. pachyptila is found in areas with a high input of hydrothermal fluid, and therefore with a high [CO₂]. R. piscesae is found in a range of habitats with low to high levels of hydrothermal fluid input, with a correspondingly broad range of CO₂ concentrations. We examined the strategies for dissolved inorganic carbon (DIC) use by the symbionts from these two species. R. pachyptila were collected from the East Pacific Rise (9°50′N; 104°20′W) in March 1996, and R. piscesae were collected from the Juan de Fuca Ridge (47°57′N; 129°07′W) during September of 1996 and 1997. The differences in the hosts' habitats were reflected by the internal pools of DIC in these organisms. The concentrations of DIC in coelomic fluid from R. piscesae were 3.1 to 10.5 mM, lower than those previously reported for R. pachyptila, which often exceed 30 mM. When symbionts from both hosts were incubated at in situ pressures, their carbon fixation rates increased with the extracellular concentration of CO₂, and not HCO₃ ⁻, and symbionts from R. piscesae had a higher affinity for CO₂ than those from R. pachyptila (K _1/2 of 7.6 μM versus 49 μM). Transmission electron micrographs showed that symbionts from R. piscesae lack carboxysomes, irrespective of the coelomic fluid [DIC] of their host. This suggests that the higher affinity for CO₂ of R. piscesae symbionts may be their sole means of compensating for lower DIC concentrations. The δ¹³C values of tissues from R. piscesae with higher [DIC] in the coelomic fluid were more positive, opposite to the trend previously described for other autotrophs. Factors which may contribute to this trend are discussed. Received: 24 September 1998 / Accepted: 12 May 1999     

Feeding strategy and daily ration of juvenile pink shrimp (Farfantepenaeus duorarum) in a South Florida seagrass bed

 The diet of juvenile pink shrimp (Farfantepenaeus duorarum Burkenroad, previously Penaeus duorarum) from Long Key Bight, Florida Keys, was studied using stomach content examination, pigment measurements, and stable isotope (δ¹³C and δ¹⁵N) analysis. Samples were taken over approximately 24 h on four occasions from December 1997 to June 1998. Juvenile F. duorarum fed nocturnally, the main prey being the seagrass shrimp Thor floridanus (Decapoda: Caridea: Hippolytidae), which accounted for 34% of the stomach content volume. Other common components of the diet were bivalves (mainly Tellina sp.) with 15% volume, calcareous algae (8%), plant detritus (5%), copepods (3%), and seagrass fragments (2%). Pigment concentrations (chlorophyll a plus phaeopigments) in F. duorarum stomachs ranged from 7 to 73 mg l⁻¹ or 40 to 310 ng stomach⁻¹. The exponential gastric evacuation rate was determined experimentally at 1.3 ± 0.5 h⁻¹. Daily rations (in percent body weight) calculated from time series of stomach fullness ranged between 11 and 16% d⁻¹. Total consumption by the population (in wet weight) ranged between 0.05 and 0.3 g m⁻² d⁻¹. Stable isotope measurements confirmed that T. floridanus was the main food source for F. duorarum. δ¹³C-values of whole animals of both species were identical at −10.0 ± 1.6‰ PDB. δ¹⁵N-values of both species were also not significantly different (pooled mean: 5.9 ± 1.7‰). Stomach contents of wild-caught F. duorarum and stomach contents of F. duorarum fed T. floridanus also showed similar stable isotope values. Received: 12 August 1999 / Accepted: 21 March 2000     

Localisation of biogenic amines in larvae of Bugula neritina (Bryozoa: Cheilostomatida) and their effects on settlement

Distributions of serotonin and catecholamines in larvae of the marine bryozoan Bugula neritina (Bryozoa: Cheilostomatida) were investigated using immunohistochemistry with anti-serotonin antiserum and glyoxylic acid–induced fluorescence histochemistry. Anti-serotonin immunoreactive substances and glyoxylic acid–induced fluorescent substances had similar distributions in the equatorial neuromuscular ring, the neural plexus, the paired axial neuromuscular cords, and tracts connecting the neural plexus to ciliated cells bordering the pyriform organ. The effects of dopamine, noradrenaline, adrenaline, tyramine, octopamine, synephrine and serotonin, at 10⁻⁴, 10⁻⁵ and 10⁻⁶  M, on settlement were analysed. In filtered seawater, 98% of larvae settled in 3 h, but only 11%, 3% and 6% total settlement was observed after 8 h in 10⁻⁴  M dopamine, 10⁻⁴  M serotonin and 10⁻⁵  M serotonin, respectively. Total settlement was 70% in 10⁻⁴  M noradrenaline, 80% in 10⁻⁴  M adrenaline and 60% in 10⁻⁴  M tyramine. Less than 60% settlement was observed in 10⁻⁴ and 10⁻⁵  M octopamine and synephrine. Serotonin's inhibitory effect on settlement was mimicked by a range of serotonin receptor agonists and antagonists, among which 5-carboxamidotryptamine was the most potent. Received: 19 March 1999 / Accepted: 11 October 1999