Feeding characteristics and molecular phylogeny of the thecate mixotrophic dinoflagellate Fragilidium mexicanum

Prey spectrum and feeding process of the mixotrophic thecate dinoflagellate Fragilidium mexicanum strain Fm-LOHABE01 were examined using a culture isolated from Masan Bay, Korea in 2011 during a summer bloom of the toxic dinoflagellate Alexandrium pacificum. The novel 18S and 28S rDNA sequences for F. mexicanum were also used to explore inter-species relationships within the genus Fragilidium. The F. mexicanum fed on species belonging to four dinoflagellate genera (i.e., Alexandrium, Ceratium, Heterocapsa, and Scrippsiella) when separately offered a variety of prey, including dinoflagellates, raphidophytes, cryptophytes, and a ciliate. In addition, F. mexicanum displayed different levels of feeding frequency for prey species of Alexandrium. While F. mexicanum consistently fed on A. catenella and A. pacificum, feeding on A. affine was rarely observed. The F. mexicanum ingested prey by direct engulfment through the sulcus, after capturing the prey by a tow filament. Phylogenetic analyses of 18S and 28S rDNA datasets demonstrated that Fragilidium sequences formed a monophyletic group with high statistical supports and diverged into four distinct clades. The F. mexicanum formed a separate clade with Fragilidium sp. EUSK D from Angola and Korean isolate of F. fissile with very strong supports.     

Tropical Forest Fragmentation Affects Floral Visitors but Not the Structure of Individual-Based Palm-Pollinator Networks

Despite increasing knowledge about the effects of habitat loss on pollinators in natural landscapes, information is very limited regarding the underlying mechanisms of forest fragmentation affecting plant-pollinator interactions in such landscapes. Here, we used a network approach to describe the effects of forest fragmentation on the patterns of interactions involving the understory dominant palm Astrocaryum mexicanum (Arecaceae) and its floral visitors (including both effective and non-effective pollinators) at the individual level in a Mexican tropical rainforest landscape. Specifically, we asked: (i) Does fragment size affect the structure of individual-based plant-pollinator networks? (ii) Does the core of highly interacting visitor species change along the fragmentation size gradient? (iii) Does forest fragment size influence the abundance of effective pollinators of A. mexicanum? We found that fragment size did not affect the topological structure of the individual-based palm-pollinator network. Furthermore, while the composition of peripheral non-effective pollinators changed depending on fragment size, effective core generalist species of pollinators remained stable. We also observed that both abundance and variance of effective pollinators of male and female flowers of A. mexicanum increased with forest fragment size. These findings indicate that the presence of effective pollinators in the core of all forest fragments could keep the network structure stable along the gradient of forest fragmentation. In addition, pollination of A. mexicanum could be more effective in larger fragments, since the greater abundance of pollinators in these fragments may increase the amount of pollen and diversity of pollen donors between flowers of individual plants. Given the prevalence of fragmentation in tropical ecosystems, our results indicate that the current patterns of land use will have consequences on the underlying mechanisms of pollination in remnant forests.     

Vitellogenin synthesis and characterisation of the liver estrogen receptor in the neotenous salamander Ambystoma mexicanum

The neotenous salamander Ambystoma mexicanum reaches sexual maturity without completing metamorphosis. Females must, therefore, synthesize vitellogenin, the precursor of the egg-yolk proteins. We show that livers of female axolotls synthesize and secrete a phosphoprotein which migrates with Xenopus vitellogenin on SDS-gels and is precipitated by antibody prepared against Xenopus vitellogenin. The livers of male axolotls do not normally synthesize this protein but can be induced to do so by treatment in vivo with estradiol. A receptor with a high affinity for estradiol (K_d = 0.3 × 10^?9M) was found in the nuclei prepared from livers of male and female axolotls. It sediments at 3.7 S at 0°C in sucrose gradients containing 0.5 M KCl. Each nucleus contains about 1300 binding sites for estradiol, 13 times the number found in normal male Xenopus nuclei, but as axolotl nuclei are about 12 times larger, the concentrations of binding sites are similar. In contrast to Xenopus, there is no detectable increase in the number of nuclear binding sites following estrogen treatment. We conclude that the controls affecting both the appearance of vitellogenin inducibility and the induction of vitellogenin synthesis differ between the two species A. mexicanum and Xenopus laevis.     

Myosin polymorphism in urodelan amphibian dorsal skeletal muscle

• 1.1. Myosin isoforms were analyzed in the dorsal skeletal muscle of four urodelan amphibian species using a modified pyrophosphate gel electrophoresis which allowed better discrimination than classical methods.
• 2.2. The three fast and the intermediate isomyosins were characterized by a specific heavy chain, respectively HCf and HCi, associated with different combinations of the fast light chains LC1f, LC2f and LC3f.
• 3.3. Slow myosin was characterized by one (P. waltlii, T. palmatus, S. maculata) or two (T. alpestris) isoforms, combining a specific slow myosin heavy chain (HCs) with slow light chains only in the case of P. waltlii, or with slow and fast light chains in the other species.

     

Regulation by thyroid hormones of terminal differentiation in the skeletal dorsal muscle. II. Urodelan amphibians

In the urodelan amphibian Pleurodeles waltlii, spontaneous anatomical metamorphosis was correlated with an increase in the serum level of thyroxine (T4). It was also accompanied by a change in the myofibrillar ATPase profile of the dorsal skeletal muscle; fibers of larval type were gradually replaced by the adult fiber types I, II A, and II B. Likewise, a myosin isoenzymic transition was observed in dorsal muscle, larval isomyosins were replaced by adult isoforms. In a related species, Ambystoma mexicanum, in which no spontaneous external metamorphosis occurs under standard conditions, the serum T4 level was shown to remain low. During further development, the myofibrillar ATPase profile acquired the adult fiber types, but a high percentage of immature fibers of type II C persisted. Myosin isoenzymic transition was also incomplete; larval isoforms were still distinguished in the neotenic adults. In experimental hypothyroidian P. waltlii, no external metamorphosis occurred; the myofibrillar ATPase profile was of the immature type, and the larval isomyosins persisted. Triiodothyronine induced experimental anatomical metamorphosis in A. mexicanum; only limited changes in the myofibrillar ATPase profile resulted from the treatment, but a complete myosin isoenzymic transition was observed. These results tend to indicate that a moderate increase in the level of thyroid hormone is sufficient to induce the differentiation of adult fiber types, together with the production of adult myosin isoforms in the skeletal dorsal muscle of amphibians, while a pronounced increase would be necessary for repressing the initial larval features.     

Variation in Salamander Tail Regeneration Is Associated with Genetic Factors That Determine Tail Morphology

Very little is known about the factors that cause variation in regenerative potential within and between species. Here, we used a genetic approach to identify heritable genetic factors that explain variation in tail regenerative outgrowth. A hybrid ambystomatid salamander (Ambystoma mexicanum x A. andersoni) was crossed to an A. mexicanum and 217 offspring were induced to undergo metamorphosis and attain terrestrial adult morphology using thyroid hormone. Following metamorphosis, each salamander’s tail tip was amputated and allowed to regenerate, and then amputated a second time and allowed to regenerate. Also, DNA was isolated from all individuals and genotypes were determined for 187 molecular markers distributed throughout the genome. The area of tissue that regenerated after the first and second amputations was highly positively correlated across males and females. Males presented wider tails and regenerated more tail tissue during both episodes of regeneration. Approximately 66–68% of the variation in regenerative outgrowth was explained by tail width, while tail length and genetic sex did not explain a significant amount of variation. A small effect QTL was identified as having a sex-independent effect on tail regeneration, but this QTL was only identified for the first episode of regeneration. Several molecular markers significantly affected regenerative outgrowth during both episodes of regeneration, but the effect sizes were small (<4%) and correlated with tail width. The results show that ambysex and minor effect QTL explain variation in adult tail morphology and importantly, tail width. In turn, tail width at the amputation plane largely determines the rate of regenerative outgrowth. Because amputations in this study were made at approximately the same position of the tail, our results resolve an outstanding question in regenerative biology: regenerative outgrowth positively co-varies as a function of tail width at the amputation site.     

A strong conditional mutualism limits and enhances seed dispersal and germination of a tropical palm

Seed predation and seed dispersal can have strong effects on early life history stages of plants. These processes have often been studied as individual effects, but the degree to which their relative importance co-varies with seed predator abundance and how this influences seed germination rates is poorly understood. Therefore, we used a combination of observations and field experiments to determine the degree to which germination rates of the palm Astrocaryum mexicanum varied with abundance of a small mammal seed predator/disperser, Heteromys desmarestianus, in a lowland tropical forest. Patterns of abundance of the two species were strongly related; density of H. desmarestianus was low in sites with low density of A. mexicanum and vice versa. Rates of predation and dispersal of A. mexicanum seeds depended on abundance of H. desmarestianus; sites with high densities of H. desmarestianus had the highest rates of seed predation and lowest rates of seed germination, but a greater total number of seeds were dispersed and there was greater density of seedlings, saplings, and adults of A. mexicanum in these sites. When abundance of H. desmarestianus was experimentally reduced, rates of seed predation decreased, but so did dispersal of A. mexicanum seeds. Critically, rates of germination of dispersed seeds were 5 times greater than undispersed seeds. The results suggest that the relationship between A. mexicanum and H. desmarestianus is a conditional mutualism that results in a strong local effect on the abundance of each species. However, the magnitude and direction of these effects are determined by the relative strength of opposing, but related, mechanisms. A. mexicanum nuts provide H. desmarestianus with a critical food resource, and while seed predation on A. mexicanum nuts by H. desmarestianus is very intense, A. mexicanum ultimately benefits because of the relatively high germination rates of its seeds that are dispersed by H. desmarestianus.     

MORPHOLOGY AND TAXONOMY OF PROROCENTRUM MEXICANUM AND REINSTATEMENT OF PROROCENTRUM RHATHYMUM (DINOPHYCEAE)1

Dinoflagellates collected during red tide events in Bahia Mazatlan, Mexico during the early spring of 1999 and 2000 appeared under LM to belong to Prorocentrum mexicanum Osorio‐Tafall. Observations with SEM of those populations showed marked differences in shape and microornamentation from the related species, Prorocentrum rhathymum Loeblich III, Sherley and Schmidt. In P. mexicanum, the presence and dimensions of poroids, the uneven distribution of trichocyst pores not located in depressions, and the general architecture of the periflagellar region are more closely related to Prorocentrum caribbaeum Faust. Also, P. mexicanum has a three‐horned (sometimes two‐horned) spine and is deeper in the anterior than the posterior region, whereas P. rhathymum has a simple small spine and its sagittal view is oval. Furthermore, the number and distribution of trichocyst pores in the periflagellar area is different between the two species, being located on both valves in P. mexicanum and only on the right valve in P. rhathymum. To date, true P. mexicanum has been described only from plankton sampling, whereas P. rhathymum was frequently mentioned associated with floating detritus (macroalgae) but also forming red tides. Altogether, the evidence presented demonstrates that P. mexicanum (planktonic) and P. rhathymum (epibenthic) are distinct species and are not synonyms, as is often accepted.     

Changes in amino acid uptake and protein synthesis of bullfrog tadpole liver and tail tissues during triiodothyronine-induced metamorphosis

The effect of triiodothyronine (T₃′) on the uptake of several amino acids into the amino acid pools and into proteins of Rana catesbeiana tadpole liver and tail muscle and tail fin has been studied. Labeling of the alanine and glycine pool was stimulated in the liver more than the leucine pool. After exposure to T₃ for 3 days, uptake of α-aminoisobutyric acid (a transport model substrate) into liver was stimulated about 55%. In tail tissues uptake of leucine was stimulated but uptake of alanine was depressed by T₃. Incorporation of leucine and alanine into tissue protein was stimulated in the liver but inhibited in tail tissues after T₃ injection.Changes in other macromolecules and ATP and ADP levels in liver and tail muscle were also investigated during induced metamorphosis. In the liver, the total DNA content did not change, but the RNA and protein content per liver increased significantly. The increase in RNA/DNA and protein/DNA ratios, suggested that liver cells underwent hypertrophy during induced metamorphosis. The ATP level showed a transient decrease after 3 days of T₃ treatment. In tail muscle, protein and RNA content decreased as the muscle regressed, but the DNA content and ATP level remained unchanged throughout the experimental period.     

Genie Control of Axolotl Metamorphosis

SYNOPSIS. Neoteny in the Mexican axolotl, Ambystoma mexicanum,is caused by homozygosity for a single recessive gene. The dominantallele causing physical metamorphosis is found in the closelyrelated species, Ambystoma tigrinum, with which it can hybridize.Despite the failure of axolotls to undergo physical metamorphosis,they do undergo a cryptic metamorphosis. A larval-to-adult hemoglobinform change, serum protein changes and other physiological eventsusually associated with amphibian metamorphosis occur duringearly larval life at ages comparable to the age at which Ambystomatigrinum undergoes both the cryptic and external metamorphicevents. Axolotl cryptic metamorphosis can be induced precociouslyby immersion of the larvae in low concentrations of thyroxine;physical metamorphosis can be induced with higher thyroxineconcentrations. The site of action of the gene responsible foraxolotl neoteny has not been identified. A change in the sensitivityof external metamorphic processes to thyroxine, or reduced hormonalstimulation by the pituitary or hypothalamus may be responsible.A comparison of these functions in Ambystoma tigrinum and theaxolotl may identify the lesion.     

Cell surface proteins in the early embryogenesis of Pleurodeles waltlii

Surface proteins in the first embryonic stages (8–32 cells, morula, blastula, early and late gastrula) of Pleurodeles waltlii were selectively labelled by ¹²⁵I using lactoperoxidase and glucose/glucose oxidase. Iodination was effected either on non-dissociated embryos or after their dissociation with EDTA. On the outer surface of non-dissociated embryos the two-dimensional electrophoresis revealed only three groups of ¹²⁵I-labelled proteins which did not change during all studied stages. Quite different results were obtained with the cells of dissociated embryos. In addition to the iodinated proteins of the embryonic outer surface seven major iodinated proteins were identified. These proteins originate from the regions of cell-cell contacts in intact embryo. Their two-dimensional pattern in dissociated cells changes between stages 8–32 cells and morula. The next important difference was observed during gastrulation, which corresponds in Pleurodeles waltlii to the first morphogenetic movements. Therefore the outside and inside cell surfaces of embryo are different already at stage 8–32 cells (and probably earlier), before the first step of morphogenesis. The changes of cell surface proteins at early embryonal development take place inside the embryo, in the regions of cell-cell interactions.     

Nucleocytoplasmic interactions during ontogenesis in individuals obtained by intra- and interspecific nuclear transplantation in the genus Pleurodeles (urodele amphibian)

Nuclei derived from a single blastula of Pleurodeles waltlii or Pleurodeles poireti are transplanted to the cytoplasm of eggs of both species (P. waltlii and P. poireti). Animals with identical genomes are obtained, some resulting from an intraspecific nucleocytoplasmic association, the others from an interspecific association (nucleocytoplasmic hybrids). A marked lethality results from the heterospecific association. Morphological and physiological effects, concomitant to initial egg conditioning, occur during the first stages of development. The study of lactic dehydrogenase (LDH) and malic dehydrogenase (MDH) zymograms reveals the genomic activity in the embryo from stage 28 (heart beat). A regionalization of this activity is evident; the nuclear activity can be shown in the ectomesodermic tissue sooner than in the endodermal cells. From stage 38 (feeding), the functional substitution of transplanted nucleus for effects of initial cytoplasmic conditioning appears to be total. No cytoplasmic heredity can be shown: the genetic effect of the transplanted nucleus is dominant. This is corroborated by the results of cutaneous cross-grafts between adult allocytoplasmic twins. Those animals present strictly identical immune reactions; their antigenic constitution seems to be independent from the specific origin of the egg cytoplasm.     

Comparison of the NAD Kinase and Myosin Light Chain Kinase Activator Properties of Vertebrate, Higher Plant, and Algal Calmodulins

In the preceding paper (Lukas, Iverson, Schleicher, Watterson 1984 Plant Physiol 75: 788-795), we reported that the amino acid sequence of spinach calmodulin has at least 13 amino acid sequence differences from vertebrate calmodulin. In the present study, we investigated the effect of these amino acid sequence substitutions on the enzyme activator properties of vertebrate and plant calmodulins. Calmodulins from spinach and the green alga Chlamydomonas reinhardtii activate chicken gizzard myosin light chain kinase in a manner similar but not identical to chicken calmodulin. In contrast, these calmodulins have very different NAD kinase activator properties. The concentration required for half-maximal activation of pea seedling NAD kinase by spinach calmodulin (3-4 nanomolar) is lower than the corresponding concentrations of chicken (20 nanomolar) and Chlamydomonas (40 nanomolar) calmodulins. However, the maximum level of activation obtained with Chlamydomonas calmodulin is 4- to 6-fold higher than spinach or chicken calmodulin. These data indicate that the limited structural heterogeneity among calmodulins have differential effects on their biochemical activities.     

Spiny lobster development: where does successful metamorphosis to the puerulus occur?: a review

This review re-addresses the question: Where does metamorphosis to the puerulus mainly take place among the shallow-water palinurids? A decade ago we reviewed this ecological question in a paper that focused on phyllosomal development of the western rock lobster, Panulirus cygnus. The main region of occurrence of its metamorphosis was found to be in the slope region beyond the shelf break. Because the puerulus of P. cygnus is a non-feeding stage, it was hypothesised that metamorphosis will not occur until the final phyllosoma has reached some critical, and specific, level of stored energy reserves. For late larval development and successful metamorphosis of P. cygnus, the richest food resources seem to be located in the slope waters adjoining the shelf break off Western Australia. This, like most shelf break areas, is a region of higher zooplankton and micronekton biomass than is usually found further offshore, and is dominated (in winter-spring months) by the warm south-flowing Leeuwin Current. In this new review, distribution and abundance data of final phyllosomas and pueruli are examined from, Panulirus argus, Panulirus cygnus, Panulirus japonicus, Panulirus ornatus and Jasus edwardsii, and where possible, related to features of the satellite imagery of the areas in which they occur. We hypothesise that metamorphosis will occur where the final stages have partaken of sufficient, appropriate nutrition to provide them with a reserve of bioenergetic resources, and this can occur where oceanographic fronts effect greater planktonic productivity and concentrations of food organisms. This may be near the shelf-break, or out to large distances offshore, because of large-scale oceanographic events such as the prevailing current system, its off-shoots, mesoscale eddy fronts, counter-currents, etc. However, we contend that, in terms of population recruitment, metamorphosis in most shallow-water palinurid species occurs mainly in the slope waters adjoining the shelf break of the region to which the species is endemic. Although some final phyllosomas may metamorphose much further offshore, it is unlikely that these pueruli will reach the shore, let alone settle and successfully moult to the juvenile stage. All of the data indicate that successful metamorphosis from the final-stage phyllosoma to the puerulus stage in all species occurs offshore but close to the continental shelf.     

Immuno-electron microscopic localization of calmodulin and calmodulin-binding proteins in the mouse germ cells during spermatogenesis and maturation

When extracts of mouse testis were Western-blotted against a monoclonal antibody which reacts with calmodulin in the presence of Ca²⁺, all calmodulin was associated with the macromolecules of molecular weight above 50 kDa. Immuno-electron microscopy of testes using this antibody indicated that calmodulin is localized at higher density in the nucleus and cytoplasm of germ cells during the developmental phase between pachytene and round spermatid, showing the highest level just before meiotic divisions. There was no special association of calmodulin to any organelles in these cells. Extremely low levels of calmodulin occurred in spermatogonia and other testicular tissue cells. Calmodulin decreased dramatically as spermatids underwent metamorphosis, becoming detectable only at the perinuclear space of sperm heads. Further relocation to the postacrosomal region occurred during sperm transit to the cauda epididymis. Immunodetection after the calmodulin overlay on ultrathin sections revealed a sharp increase of calmodulin immunogold deposits in the nuclei of spermatids accompanying their condensation. The results indicate that some calmodulin-binding proteins, but not calmodulin itself, accumulate in the nuclei during the final steps of spermiogenesis.     

Metamorphosis of a Scleractinian Coral in Response to Microbial Biofilms

Microorganisms have been reported to induce settlement and metamorphosis in a wide range of marine invertebrate species. However, the primary cue reported for metamorphosis of coral larvae is calcareous coralline algae (CCA). Herein we report the community structure of developing coral reef biofilms and the potential role they play in triggering the metamorphosis of a scleractinian coral. Two-week-old biofilms induced metamorphosis in less than 10% of larvae, whereas metamorphosis increased significantly on older biofilms, with a maximum of 41% occurring on 8-week-old microbial films. There was a significant influence of depth in 4- and 8-week biofilms, with greater levels of metamorphosis occurring in response to shallow-water communities. Importantly, larvae were found to settle and metamorphose in response to microbial biofilms lacking CCA from both shallow and deep treatments, indicating that microorganisms not associated with CCA may play a significant role in coral metamorphosis. A polyphasic approach consisting of scanning electron microscopy, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE) revealed that coral reef biofilms were comprised of complex bacterial and microalgal communities which were distinct at each depth and time. Principal-component analysis of FISH data showed that the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Cytophaga-Flavobacterium of Bacteroidetes had the largest influence on overall community composition. A low abundance of Archaea was detected in almost all biofilms, providing the first report of Archaea associated with coral reef biofilms. No differences in the relative densities of each subdivision of Proteobacteria were observed between slides that induced larval metamorphosis and those that did not. Comparative cluster analysis of bacterial DGGE patterns also revealed that there were clear age and depth distinctions in biofilm community structure; however, no difference was detected in banding profiles between biofilms which induced larval metamorphosis and those where no metamorphosis occurred. This investigation demonstrates that complex microbial communities can induce coral metamorphosis in the absence of CCA.     

Immunocytochemical localization and spatial relation to the adenohypophysis of a somatostatin-like and a corticotropin-releasing factor-like peptide in the brain of four amphibian species

Summary The distribution of somatostatin (SRIF) — and corticotropin-releasing factor (CRF)-like — immunoreactive material was studied in the brain of four amphibian species (Ambystoma mexicanum, Pleurodeles waltlii, Xenopus laevis, Rana ridibunda) by use of immunocytochemistry. A wide network of SRIF-immunoreactive fibers and numerous perikarya were observed in all amphibians examined, with a dense accumulation of nerve endings in the external layer of the median eminence (ELME). In the representatives of the four amphibian species the CRF-like system was more circumscribed. Immunoreactive perikarya were present in the preoptic area, mainly in a ventrobasal position, and in the interpeduncular nucleus. The tract running along the ventral part of the tuber cinereum ends in the ELME facing the rostroventral lobe of the pars distalis that contains corticotrophs. CRF fibers were scarce or absent in the neural lobe. In all species studied in the present work, CRF fibers end in the area of the ELME close to the pituitary lobe containing corticotrophs. This correlation is similar to that reported for the Japanese quail and several teleosts.This work was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek and the CNRS