Isolation and characterization of eight polymorphic microsatellite loci in the endangered Hawaiian tree snail, Achatinella sowerbyana.

Achatinella is a genus of highly endangered native Hawaiian tree snails. Eight polymorphic microsatellite markers were isolated for one species, Achatinella sowerbyana, to be used in assessing remaining genetic diversity and gene flow in the often small and isolated populations. All loci amplified consistently in every individual tested. The number of alleles per locus in a sample of 40 individuals varied from three to 13, and the observed heterozygosity ranged from 0.308 to 0.846. These markers will be used to assist in the development of conservation and management plans.

Biofouling likely serves as a major mode of dispersal for the polychaete tubeworm Hydroides elegans as inferred from microsatellite loci.

The polychaete tubeworm Hydroides elegans (Haswell) is a biofouling species with relatively limited larval dispersal. Four highly polymorphic microsatellite loci were used to make inferences about the migration and global population structure of 137 individuals from seven sub-populations located in the Atlantic, Pacific, and Indian Oceans and in the Mediterranean Sea. The results of the genetic analyses suggest minimal population sub-structure (F(st) = 0.09). Estimates of pairwise F(st) and migration rates using the coalescent-based method of MIGRATE suggest that there is little genetic differentiation between certain populations. Variation in relatedness among pairs of populations is consistent with a suite of local and global factors. The most likely explanation for close genetic relatedness among certain populations over such vast distances is the regular and consistent transport of adults and larvae on the hulls and in the ballast water of ships, respectively.

What is metamorphosis?

Metamorphosis (Gr. meta- "change" + morphe "form") as a biological process is generally attributed to a subset of animals: most famously insects and amphibians, but some fish and many marine invertebrates as well. We held a symposium at the 2006 Society for Integrative and Comparative Biology (SICB) annual meeting in Orlando, FL (USA) to discuss metamorphosis in a comparative context. Specifically, we considered the possibility that the term "metamorphosis" could be rightly applied to non-animals as well, including fungi, flowering plants, and some marine algae. Clearly, the answer depends upon how metamorphosis is defined. As we participants differed (sometimes quite substantially) in how we defined the term, we decided to present each of our conceptions of metamorphosis in 1 place, rather than attempting to agree on a single consensus definition. Herein we have gathered together our various definitions of metamorphosis, and offer an analysis that highlights some of the main similarities and differences among them. We present this article not only as an introduction to this symposium volume, but also as a reference tool that can be used by others interested in metamorphosis. Ultimately, we hope that this article-and the volume as a whole-will represent a springboard for further investigations into the surprisingly deep mechanistic similarities among independently evolved life cycle transitions across kingdoms.

Histochemical survey of transmitters in the central ganglia of the gastropod mollusc Phestilla sibogae.

The aeolid nudibranch Phestilla sibogae is well studied in terms of its larval nervous system and neuronal involvement in metamorphosis. Central neurones in the adult have also been identified anatomically and electrophysiologically. We describe the neurotransmitter contents of these neurones and provide details of neuritic projections and developmental changes during growth (3 to 18 mm body length). Central ganglia from specimens of all sizes contained 100-115 serotonin-immunoreactive neurones, some of which appeared to be homologues of cells identified in other gastropods. Tyrosine hydroxylase immunoreactivity and aldehyde-induced fluorescence marked a common set of 28-30 catecholaminergic neurones located anteriorly in the cerebropleural ganglia and laterally in the pedal ganglia. Ganglionic neuropile and nerve trunks also contained many catecholaminergic fibres. About 65-100 intensely labelled FMRFamide-immunoreactive neurones were located symmetrically throughout the central ganglia, although one population was located only in the right pedal ganglion. Another 40-45 FMRFamide-immunoreactive neurones were weakly or variably stained. Central ganglia also contained 27-29 intensely labelled pedalpeptide-immunoreactive neurones, including those that were apparently homologues of cells previously described in Tritonia diomedea, and 16-19 weakly labelled pedal-peptide-immunoreactive neurones, including giant cerebropleural neurones coexhibiting FMRFamide immunoreactivity. Little cell addition involving any transmitter phenotype occurred as animals grew in body length, body growth being accommodated by growth in the size of individual cells, consistent with an approximate doubling in the size of the ganglia themselves.

Larval muscle contraction fails to produce torsion in a trochoidean gastropod.

The causes and effects of ontogenetic torsion in gastropods have been debated intensely for more than a century (1-19). Occurring rapidly and very early in development, torsion figures prominently in shaping both the larval and adult body plans. We show that mechanical explanations of the ontogenetic event that invoke contraction of larval retractor muscles are inadequate to explain the observed consequences in some gastropods. The classic mechanical explanation of Crofts (4, 5) and subsequent refinements of her explanation have been based on species with rigid larval shell properties (18, 19) that cannot be extrapolated to all gastropods. We present visual evidence of the lack of rigidity of the uncalcified larval shell in a basal trochid gastropod, Margarites pupillus (Gould), and provide photographic confirmation of our prediction that larval retractor muscle contraction is insufficient to produce more than local deformation or dimpling at the site of muscle insertion. These findings do not refute muscular contraction as a primary cause of ontogenetic torsion in gastropods that calcify their larval shells prior to the onset of torsion, nor do they refute the monophyly of torsion. They do, however, suggest that torsion may be a loosely constrained developmental process with multiple pathways to the more constrained end result (20, 21).

Mitochondrial phylogeny of extant Hawaiian tree snails (Achatinellinae).

Hawaiian tree snails in the endemic subfamily Achatinellinae display a staggering variety of shell colors and banding patterns. Despite numerous attempts to classify this morphological variation, a conclusive phylogeny has not been proposed. To improve conservation efforts, we sought to better understand the species identities and phylogenetic relationships among the extant species of Achatinella and Partulina using partial mitochondrial 16S ribosomal DNA sequences. The reconstructed phylogeny showed a high degree of support for more recent branches, but gave little support to deeper nodes. The most confident branches challenge previous systematic arrangements of these snails, grouping species that previously had been placed into different subgenera. High levels of sequence divergence within some species may reflect the long-term isolation of subpopulations. Rapid rates of sequence divergence may have saturated base substitutions and contributed to the lack of resolution of higher-order relationships. We did not find support for the monophyly of the Achatinella species, nor thus for a single colonization of Oahu from Maui Nui.

Catecholamines modulate metamorphosis in the opisthobranch gastropod Phestilla sibogae.

Larvae of the nudibranch Phestilla sibogae are induced to metamorphose by a factor from their adult prey, the coral Porites compressa. Levels of endogenous catecholamines increase 6 to 9 days after fertilization, when larvae become competent for metamorphosis. Six- to nine-day larvae, treated with the catecholamine precursor L-DOPA (0.01 mM for 0.5 h), were assayed for metamorphosis in response to coral inducer and for catecholamine content by high-performance liquid chromatography. L-DOPA treatment caused 20- to 50-fold increases in dopamine, with proportionally greater increases in younger larvae, so that L-DOPA-treated larvae of all ages contained similar levels of dopamine. A much smaller (about twofold) increase in norepinephrine occurred in all larvae. The treatment significantly potentiated the frequency of metamorphosis of 7- to 9-d larvae at low concentrations of inducer. In addition, L-DOPA treatment at 9 d increased aldehyde-induced fluorescence in cells that were also labeled in the controls, and revealed additional cells. However, all labeled cells were consistent with the locations of cells showing tyrosine-hydroxylase-like immunoreactivity. Catecholamines are likely to modulate metamorphosis in P. sibogae, but rising levels of catecholamines around the time of competence are insufficient alone to account for sensitivity to inducer in competent larvae.

Ewing's family of tumors involving structures related to the central nervous system: a review.

This review consolidates information gleaned from several case reports and larger series on Ewing's sarcoma family of tumors (EFT) involving structures related to and found in the central nervous system (CNS). These tumors involve the skull, the spinal column, adjacent soft tissues, the meninges, and the brain. We have separated the cases by skull region and spinal column level, and we discuss the attendant differences in prognosis following treatment by neurosurgery, radiation, and chemotherapy. Light and electron microscopic features can be used to differentiate EFT from other small round blue cell tumors that involve the CNS (central primitive neuroectodermal tumor, lymphoma, etc.). Recent molecular and genetic findings in EFT provide new diagnostic methods. We conclude that EFT involving the CNS and adjacent structures is not so rare as previously stated and that the prognosis is more favorable, as a rule, than for the more common examples arising in the long bones and pelvis.

The apical sensory organ of a gastropod veliger is a receptor for settlement cues.

On the basis of anatomy and larval behavior, the apical sensory organ (ASO) of gastropod veliger larvae has been implicated as the site of perception of cues for settlement and metamorphosis. Until now, there have been no experimental data to support this hypothesis. In this study, cells in the ASO of veliger larvae of the tropical nudibranch Phestilla sibogae were stained with the styryl vital dye DASPEI and then irradiated with a narrow excitatory light beam on a fluorescence microscope. When its ASO cells were bleached by irradiation for 20 min or longer, an otherwise healthy larva was no longer able to respond to the usual metamorphic cue, a soluble metabolite from a coral prey of the adult nudibranch. The irradiated cells absorbed the dye acridine orange, suggesting that they were dying. When larvae not stained with DASPEI were similarly irradiated, or when stained larvae were irradiated with the light beam focused on other parts of the body, there was no loss of ability to metamorphose. Together these data provide strong support for the hypothesis. Potassium and cesium ions, known to induce metamorphosis in larvae of many marine-invertebrate phyla, continue to induce metamorphosis in larvae that have lost the ability to respond to the coral inducer due to staining and irradiation. These results demonstrate that (1) the ASO-ablated larvae have not lost the ability to metamorphose and (2) the ions do not act only on the metamorphic-signal receptor cells, but at other sites downstream in the metamorphic signal transduction pathway.

Why and how marine-invertebrate larvae metamorphose so fast.

It is argued that larviparous development has evolved at least eight times among extant animals. A 'need for speed hypothesis' is proposed to explain profound convergence on a pattern of small larvae and rapid metamorphosis across six marine invertebrate clades. Shared selection pressures include limits to larval size, the plankton-to-benthos transition, extreme hazards on the benthos, and the profound helplessness of metamorphosing animals. The adaptive mechanisms include: (1) development of juvenile structures in larvae before they are metamorphically competent; (2) external cues trigger metamorphosis; and (3) rapid cell-to-cell conductance of the metamorphic signal to bring about rapid loss of larval structures and release of juvenile structures. Both pattern and mechanisms contrast in every regard with those of the other two major larviparous clades, Insecta and Amphibia.

An inducer of molluscan metamorphosis transforms activity patterns in a larval nervous system.

Larvae of the nudibranch mollusc Phestilla sibogae metamorphose in response to a small organic compound released into seawater by their adult prey, the scleractinian coral Porites compressa. The transformations that occur during metamorphosis, including loss of the ciliated velum (swimming organ), evacuation of the shell, and bodily elongation, are thought to be controlled by a combination of neuronal and neuroendocrine activities. Activation of peripheral chemosensory neurons by the metamorphosis-inducing compound should therefore elicit changes within the central nervous system. We used extracellular recording techniques in an attempt to detect responses of neurons within the larval central ganglia to seawater conditioned by P. compressa, to seawater conditioned by the weakly inductive coral Pocillopora damicornis, and to non-inductive seawater controls. The activity patterns within the nervous systems of semi-intact larvae changed in response to both types of coral exudates. Changes took place in two size classes of action potentials, one of which is known to be associated with velar ciliary arrests.

Temporal and spatial variation in the fouling of silicone coatings in Pearl Harbor, Hawaii.

An antifouling or foul-release coating cannot be globally effective if it does not perform well in a range of environmental conditions, against a diversity of fouling organisms. From 1996 to 1998, the field test sites participating in the United States Navy's Office of Naval Research 6.2 Biofouling program examined global variation in the performance of 3 silicone foul-release coatings, viz. GE RTV11, Dow Corning RTV 3140, and Intersleek (International Coatings Ltd), together with a control anticorrosive coating (Ameron Protective Coatings F-150 series). At the University of Hawaii's test site in Pearl Harbor, significant differences were observed among the coatings in the rate of accumulation of fouling. The control coating failed rapidly; after 180-220 d immersion a community dominated by molluscs and sponges developed that persisted for the remainder of the experiment. Fouling of the GE and Dow Corning silicone coatings was slower, but eventually reached a similar community structure and coverage as the control coatings. The Intersleek coating remained lightly fouled throughout the experiment. Spatial variation in the structure of the community fouling the coatings was observed, but not in the extent of fouling. The rate of accumulation of fouling reflected differences among the coatings in adhesion of the tubeworm Hydroides elegans. The surface properties of these coatings may have affected the rate of fouling and the structure of the fouling community through their influence on larval settlement and subsequent interactions with other residents, predators, and the physical environment.

Cellular and subcellular structure of anterior sensory pathways in Phestilla sibogae (Gastropoda, Nudibranchia).

Two sensory-cell types, subepithelial sensory cells (SSCs) and intraepithelial sensory cells (ISCs), were identified in the anterior sensory organs (ASO: pairs of rhinophores and oral tentacles, and the anterior field formed by the oral plate and cephalic shield) of the nudibranch Phestilla sibogae after filling through anterior nerves with the neuronal tracers biocytin and Lucifer Yellow. A third type of sensory cells, with subepithelial somata and tufts of stiff-cilia (TSCs, presumably rheoreceptors), was identified after uptake of the mitochondrial dye DASPEI. Each sensory-cell type has a specific spatial distribution in the ASO. The highest density of ISCs is in the oral tentacles (approximately 1,200/mm2), SSCs in the middle parts of the rhinophores (>4,000/mm2), and TSCs in the tips of cephalic tentacles (100/mm2). These morphologic data, together with electrophysiologic evidence for greater chemical sensitivity of the rhinophores than the oral tentacles (Murphy and Hadfield [1997] Comp. Biochem. Physiol. 118A:727-735; Boudko et al. [1997] Soc. Neurosci. Abstr. 23:1787), led us to conclude that the two pairs of chemosensory tentacles serve different chemosensory functions in P. sibogae; i.e., ISCs and the oral tentacles serve contact- or short-distance chemoreception, and SSCs and the rhinophores function for long-distance chemoreception or olfaction. If this is true, then the ISC subsystem probably represents an earlier stage in the evolution and adaptations of gastropod chemosensory biology, whereas among the opisthobranchs, the SSC subsystem evolved with the rhinophores from ancestral cephalaspidean opisthobranchs.

Human brain tumors and exposure to metal and non-metal elements: a case-control study.

BACKGROUND: Primary brain tumors are among the most deadly of all cancers, with a 1-year survival rate of 52%. Certain elements, such as nickel, cadmium, chromium, arsenic, and beryllium, are established carcinogens in other organs. Silicon and titanium are suspected carcinogens and other elements are known to promote or inhibit the rate of tumor growth. Knowledge about the carcinogenicity of these elements in the brain is limited. In this study, we investigated the potential role of these elements as risk factors for human brain tumors. METHODS: In a case-control study, we assessed brain biopsies from 12 patients with various types of primary brain tumors and in tumor-free brain tissue from 6 autopsy cases. We used energy-dispersive X-ray analysis (EDX) to determine if there were significant differences in the concentration of the study elements in tumors and in control brains. RESULTS: In a bivariate analysis, a statistically significant association was observed between the presence of brain tumors and the concentrations of silicon (p = 0.01), magnesium (p = 0.01), and calcium (p = 0.03). Zinc was also associated with a borderline significance (p = 0.05). No association was observed for nickel (p = 0.74). Although the magnitude of the observed association was estimated using multiple logistic regression analyses, the relative risk estimates were imprecise because of insufficient sample size. Further research using a larger sample size is needed to elucidate the role of these elements in human brain carcinogenesis.

Metamorphic-Signal Transduction in Hydroides elegans (Polychaeta: Serpulidae) Is Not Mediated by a G Protein.

Evidence from larvae of hydrozoans, gastropods, and barnacles suggests that G protein-coupled receptors mediate induction of settlement and metamorphosis in response to environmental cues. We examined responses of larvae of the serpulid polychaete Hydroides elegans to neuropharmacological agents to determine if G protein-coupled receptors or their associated signal-transduction pathways regulated induction of metamorphosis by bacterial cues. Larvae of Hydroides elegans metamorphose rapidly and in high proportions when exposed to bacterial biofilms. Neither the G-protein activator Gpp[NH]p nor the inhibitor GDP-{beta}-S affected metamorphosis. Although the nonspecific phosphodiesterase inhibitors IBMX, theophylline, and papaverine induced larvae to metamorphose, RO-20-1724 (an inhibitor selective for cAMP-specific phosphodiesterase IV) and the cyclic nucleotide analogs db-cAMP and db-cGMP had no effect on metamorphosis. The adenylate cyclase activator forskolin inhibited responses of larvae to inductive bacterial biofilms. These apparently conflicting results may be due to side effects of IBMX, theophylline, papaverine, and forskolin on ion transport. The phorbol ester TPA, an activator of protein kinase C, also had no effect on larval metamorphosis. These experiments indicate that G protein-coupled receptors and signal transduction by the adenylate cyclase/cyclic AMP or phosphatidyl-inositol/ diacylglycerol/protein kinase C pathways are not components of the morphogenetic pathway that is directly responsible for processing metamorphic cues in H. elegans.

Stimulation of Metamorphosis in the Polychaete Hydroides elegans Haswell (Serpulidae).

The serpulid polychaete Hydroides elegans is a common, cosmopolitan warm-water biofouling organism. Competent larvae of H. elegans metamorphose rapidly after induction by marine biofilms. Only 15 min after coming in contact with the metamorphic cue, larvae have completed secretion of the primary tube; secretion of the secondary, calcareous tube begins 1.5 h after the primary tube has been deposited. Metamorphosis is characterized by disappearance of the prototroch and differentiation of the tentacular crown in the head region, the collar and thoracic membrane in the thoracic region, and the pygidium at the tip of the abdomen. These morphogenetic events were used to gauge the responses of larvae to biofilms, as well as to the artificial inducers Cs+ and K+. A maximal metamorphic response to the two ions requires exposure to different concentrations and durations, i.e., a 3-h pulse of 10 mM CsCl, or a 24-h continuous exposure to 50 mM excess KCl. The metamorphic response to Cs+ or K+ is much slower than the response to biofilms, demonstrating that the tissues respond differently to artificial inducers. The differences in the kinetics of the responses to the natural and cationic inducers suggest that the induction mechanisms are not the same. When these artificial inducers were used, some, but not all, of the metamorphosed juveniles never attached to the substratum or secreted a primary tube, probably as a result of secondary effects of the ions on processes of tube formation. The exact mechanisms by which Cs+ and excess K+ induce metamorphosis are still unclear, although we assume, as do others, that these agents act by depolarizing the membranes of excitable sensory cells and not by interacting with specific receptors.

Catecholamines and dihydroxyphenylalanine in metamorphosing larvae of the nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia).

The content of catecholamines and dihydroxyphenylalanine in larvae of the nudibranch Phestilla sibogae was analyzed by high-performance liquid chromatography with electrochemical detection. Dihydroxyphenylalanine, norepinephrine and dopamine were identified in larvae of all ages examined (5 through 12 days post-fertilization). Dihydroxyphenylalanine could be accurately quantified only in larvae of ages 8 through 12 days, when its average concentration increased from 0.62 to 6.71 x 10(-2) pmol micrograms protein-1. Between ages 5 and 12 days dopamine rose from 0.081 to 0.616 pmol microgram protein-1, and norepinephrine from 0.45 to 2.17 x 10(-2) pmol micrograms protein-1. Dihydroxyphenylalanine, dopamine and norepinephrine were also measured at different stages of metamorphic progress in 10- to 12-day larvae. Dihydroxyphenylalanine increased by a factor of 2.4 between the onset and completion of metamorphosis, but levels of dopamine and norepinephrine remained stable. One millimolar alpha-methyl-DL-m-tyrosine, an inhibitor of catecholamine synthesis, inhibited natural metamorphosis and depleted endogenous norepinephrine and especially dopamine, respectively, to 75% and 35% of control values. The existence of unexpectedly high levels of catecholamines in metamorphically competent larvae, and the association of catecholamine depletion with inhibition of metamorphosis, indicate that these compounds may participate in the control of gastropod development.

Caffeine and the olfactory bulb.

Caffeine, a popular CNS stimulant, is the most widely used neuroactive drug. Present in coffee, tea, chocolate, and soft drinks as well as over-the-counter and prescription medications, it influences millions of users. This agent has achieved recent notoriety because its dependency consequences and addictive potential have been re-examined and emphasized. Caffeine's central actions are thought to be mediated through adenosine (A) receptors and monoamine neurotransmitters. The present article suggests that the olfactory bulb (OB) may be an important site in the brain that is responsible for caffeine's central actions in several species. This conclusion is based on the extraordinarily robust and selective effects of caffeine on norepinephrine (NE), dopamine (DA), and particularly serotonin (5HT) utilization in the OB of mice. We believe that these phenomena should be given appropriate consideration as a basis for caffeine's central actions, even in primates. Concurrently, we review a rich rodent literature concerned with A, 5HT, NE, and DA receptors in the OB and related structures along with other monoamine parameters. We also review a more limited literature concerned with the primate OB. Finally, we cite the literature that treats the dependency and addictive effects of caffeine in humans, and relate the findings to possible olfactory mechanisms.

Neuro-Behçet's disease.

We are reporting a case of Bechçet's disease without vasculitis but with acute neutrophilic inflammation which involved the brain and other organs. The patient exhibited waxing and waning neurological deficits which were unresponsive to treatment. The neuroradiologic findings simulated those of multiple sclerosis. The neuropathological examination revealed an acute, focal, though disseminated encephalitis involving the frontal lobe, internal capsule, basal ganglia, cerebellum, and brainstem. The acute inflammation consisted of a neutrophilic and eosinophilic infiltration of the perivascular spaces and parenchyma without evidence of vasculitis, fibrinoid necrosis, or thrombosis. Cultures and special stains for microbial organisms were negative. Ultrastructural examination revealed no viral structures or other microorganisms. In situ hybridization for Epstein-Barr virus (EBV), herpes simplex virus type I (HSV-I), and cytomegalovirus (CMV) was negative. In this case, the lack of inflammation in the vessel walls points out that the necrotizing lesions in neuro-Bechçet's disease need not be the result of vasculitis and superimposed thrombosis, but may occur as a result of primary, acute neutrophilic inflammation.

Neuro-Behçet's disease.

We are reporting a case of Behçet's disease without vasculitis, but with acute neutrophilic inflammation which involved the brain and other organs. The patient exhibited waxing and waning neurological deficits which were unresponsive to treatment. The neuroradiologic findings simulated those of multiple sclerosis. The neuropathological examination revealed an acute, focal, though disseminated encephalitis involving the frontal lobe, internal capsule, basal ganglia, cerebellum, and brain stem. The acute inflammation consisted of a neutrophilic and eosinophilic infiltration of the perivascular spaces and parenchyma without evidence of vasculitis, fibrinoid necrosis, or thrombosis. Cultures and special stains for microbial organisms were negative. Ultrastructural examination revealed no viral structures or other microorganisms. In situ hybridization for Epstein-Barr virus (EBV), herpes simplex virus type I (HSVI), and cytomegalovirus (CMV) was negative. In this case, the lack of inflammation in the vessel walls points out that the necrotizing lesions in neuro-Behçet's disease need not be the result of vasculitis and superimposed thrombosis, but may occur as a result of primary, acute neutrophilic inflammation.