Lethal marine snow : pathogen of bivalve mollusc concealed in marine aggregates

Author Posting. © The Authors, 2005. This is the author's version of the work. It is posted here by permission of American Society of Limnology and Oceanography for personal use, not for redistribution. The definitive version was published in Limnology and Oceanography 50 (2005): 1983-1988, doi:10.4319/lo.2005.50.6.1983.We evaluated marine aggregates as environmental reservoirs for a thraustochytrid pathogen, Quahog Parasite Unknown (QPX), of the northern quahog or hard clam, Mercenaria mercenaria. Positive results from in situ hybridization and denaturing gradient gel electrophoresis confirm the presence of QPX in marine aggregates collected from coastal embayments in Cape Cod, Massachusetts, where QPX outbreaks have occurred. In laboratory experiments, aggregates were observed and recorded by entering a quahog’s pallial cavity, thereby delivering embedded particles from the water column to its benthic bivalve host. The occurrence of pathogen-laden aggregates in coastal areas experiencing repeated disease outbreaks suggests a means for the spread and survival of pathogens between epidemics and provides a specific target for environmental monitoring of those pathogens.This work was funded by an NSF grant as part of the joint NSF-NIH Ecology of Infectious Disease program, by the Woods Hole Oceanographic Institution (WHOI) Sea Grant Program, under a grant from the National Oceanic and Atmospheric Administration, U.S. Department of Commerce, and a National Science Foundation Graduate Fellowship to M. Lyons

Anteroposterior axis patterning by early canonical Wnt signaling during hemichordate development

The Wnt family of secreted proteins has been proposed to play a conserved role in early specification of the bilaterian anteroposterior (A/P) axis. This hypothesis is based predominantly on data from vertebrate embryogenesis as well as planarian regeneration and homeostasis, indicating that canonical Wnt (cWnt) signaling endows cells with positional information along the A/P axis. Outside of these phyla, there is strong support for a conserved role of cWnt signaling in the repression of anterior fates, but little comparative support for a conserved role in promotion of posterior fates. We further test the hypothesis by investigating the role of cWnt signaling during early patterning along the A/P axis of the hemichordate Saccoglossus kowalevskii. We have cloned and investigated the expression of the complete Wnt ligand and Frizzled receptor complement of S. kowalevskii during early development along with many secreted Wnt modifiers. Eleven of the 13 Wnt ligands are ectodermally expressed in overlapping domains, predominantly in the posterior, and Wnt antagonists are localized predominantly to the anterior ectoderm in a pattern reminiscent of their distribution in vertebrate embryos. Overexpression and knockdown experiments, in combination with embryological manipulations, establish the importance of cWnt signaling for repression of anterior fates and activation of mid-axial ectodermal fates during the early development of S. kowalevskii. However, surprisingly, terminal posterior fates, defined by posterior Hox genes, are unresponsive to manipulation of cWnt levels during the early establishment of the A/P axis at late blastula and early gastrula. We establish experimental support for a conserved role of Wnt signaling in the early specification of the A/P axis during deuterostome body plan diversification, and further build support for an ancestral role of this pathway in early evolution of the bilaterian A/P axis. We find strong support for a role of cWnt in suppression of anterior fates and promotion of mid-axial fates, but we find no evidence that cWnt signaling plays a role in the early specification of the most posterior axial fates in S. kowalevskii. This posterior autonomy may be a conserved feature of early deuterostome axis specification

Sexual Reproduction and Early Development in the Estuarine Sea Anemone, Nematostella vectensis Stephenson, 1935

This dissertation: 1) determines the factor(s) responsible for spawning induction in Nematostella vectensis; 2) isolates, describes, and documents the source of jelly from egg masses of N. vectensis; and 3) describes N. vectensis' early development.Nematostella vectensis were maintained on a 7-day mussel feeding/water change regime over 159 days. Within 36 hours of mussel feeding/water change, 69.1% of females and 78.5% of males spawned reliably. Through manipulation of feeding, water change, oxygen and nitrogenous waste concentrations, spawning induction was found to be triggered by the oxygen concentration associated with water change, and not by feeding. Ammonia, anemones' major waste product, inhibited this induction in a concentration-dependent manner.Female N. vectensis release eggs in a persistent jellied egg mass which is unique among the Actiniaria. The major component of this egg mass jelly was a positive periodic acid-Schiffs staining, 39.5-40.5 kD glycoprotein. Antibodies developed in rabbits against this glycoprotein bound to jelly of intact egg masses and to granules (2.8 1-1m in diameter) present in female anemone mesenteries and their associated filaments. Antibodies did not label male tissues.Nematostella vectensis embryos underwent first karyokinesis -60 minutes following the addition of sperm to eggs. Second nuclear division took place, followed by first cleavage, 90-120 minutes later. Each of the 4 blastomeres that resulted from first cleavage contained a single nucleus. Arrangement of these blastomeres ranged from radial to pseudospiral. Embryonic development was both asynchronous and holoblastic. Following formation of the 4-cell stage, 71% of embryos proceeded to cleave again to form an 8-cell stage. In each of the remaining 29% of embryos, a fusion of from 2-4 blastomeres resulted in 4 possible patterns which had no affect on either cleavage interval timing or subsequent development. The fusion event was not due to ooplasmic segregation. Blastomeres isolated from 4-celled embryos were regulative and developed into normal planula larvae and juvenile anemones that were 1/4 the size of those that developed from intact 4-celled embryos. Embryos exhibiting the fusion phenomenon were examined at the fine structural level. The fusion phenomenon resulted in formation of a secondary syncytium and was not a mere compaction of blastomeres

The Culture, Sexual and Asexual Reproduction, and Growth of the Sea Anemone Nematostella vectensis

Volume: 182Start Page: 169End Page: 17

Sperm of the Shrimp Sicyonia ingentis Undergo a Bi‐Phasic Capacitation Accompanied by Morphological Changes

Sperm removed from seminal receptacles of female Sicyonia ingentis can be induced to undergo a bi‐phasic acrosome reaction (AR), acrosomal exocytosis followed by filament formation, using egg water (EW). Sperm removed from males will not undergo any phase of the AR when incubated with EW, indicating that these sperm undergo a capacitation process after insemination. Freshly molted females (functional virgins) were placed in aquaria with males and monitored for copulation. Mated females were isolated and allowed to carry sperm for specific periods of time. At these time points, sperm were removed and assayed for the ability to undergo the AR using EW. The results indicate that sperm are competent to undergo acrosomal exocytosis after approximately 25 hr, while competency to form acrosomal filaments is not achieved until around 145 hr post‐insemination. Morphological examination of sperm removed from males and sperm removed from females revealed dramatic differences. Microscopic evidence indicates that some of the morphological changes seen during capacitation are necessary for the successful completion of the AR

DGGE-based detection method for Quahog Parasite Unknown (QPX)

Author Posting. © Inter-Research, 2006. This article is posted here by permission of Inter-Research for personal use, not for redistribution. The definitive version was published in Diseases of Aquatic Organisms 70 (2006): 115-122, doi:10.3354/dao070115.Quahog Parasite Unknown (QPX) is a significant cause of hard clam Mercenaria mercenaria mortality along the northeast coast of the United States. It infects both wild and cultured clams, often annually in plots that are heavily farmed. Subclinically infected clams can be identified by histological examination of the mantle tissue, but there is currently no method available to monitor the presence of QPX in the environment. Here, we report on a polymerase chain reaction (PCR)-based method that will facilitate the detection of QPX in natural samples and seed clams. With our method, between 10 and 100 QPX cells can be detected in 1 l of water, 1 g of sediment and 100 mg of clam tissue. Denaturing gradient gel electrophoresis (DGGE) is used to establish whether the PCR products are the same as those in the control QPX culture. We used the method to screen 100 seed clams of 15 mm, and found that 10 to 12% of the clams were positive for the presence of the QPX organism. This method represents a reliable and sensitive procedure for screening both environmental samples and potentially contaminated small clams.Quahog Parasite Unknown (QPX) is a significant cause of hard clam Mercenaria mercenaria mortality along the northeast coast of the United States. It infects both wild and cultured clams, often annually in plots that are heavily farmed. Subclinically infected clams can be identified by histological examination of the mantle tissue, but there is currently no method available to monitor the presence of QPX in the environment. Here, we report on a polymerase chain reaction (PCR)-based method that will facilitate the detection of QPX in natural samples and seed clams. With our method, between 10 and 100 QPX cells can be detected in 1 l of water, 1 g of sediment and 100 mg of clam tissue. Denaturing gradient gel electrophoresis (DGGE) is used to establish whether the PCR products are the same as those in the control QPX culture. We used the method to screen 100 seed clams of 15 mm, and found that 10 to 12% of the clams were positive for the presence of the QPX organism. This method represents a reliable and sensitive procedure for screening both environmental samples and potentially contaminated small clams

Anteroposterior axis patterning by early canonical Wnt signaling during hemichordate development

The Wnt family of secreted proteins has been proposed to play a conserved role in early specification of the bilaterian anteroposterior (A/P) axis. This hypothesis is based predominantly on data from vertebrate embryogenesis as well as planarian regeneration and homeostasis, indicating that canonical Wnt (cWnt) signaling endows cells with positional information along the A/P axis. Outside of these phyla, there is strong support for a conserved role of cWnt signaling in the repression of anterior fates, but little comparative support for a conserved role in promotion of posterior fates. We further test the hypothesis by investigating the role of cWnt signaling during early patterning along the A/P axis of the hemichordate Saccoglossus kowalevskii. We have cloned and investigated the expression of the complete Wnt ligand and Frizzled receptor complement of S. kowalevskii during early development along with many secreted Wnt modifiers. Eleven of the 13 Wnt ligands are ectodermally expressed in overlapping domains, predominantly in the posterior, and Wnt antagonists are localized predominantly to the anterior ectoderm in a pattern reminiscent of their distribution in vertebrate embryos. Overexpression and knockdown experiments, in combination with embryological manipulations, establish the importance of cWnt signaling for repression of anterior fates and activation of mid-axial ectodermal fates during the early development of S. kowalevskii. However, surprisingly, terminal posterior fates, defined by posterior Hox genes, are unresponsive to manipulation of cWnt levels during the early establishment of the A/P axis at late blastula and early gastrula. We establish experimental support for a conserved role of Wnt signaling in the early specification of the A/P axis during deuterostome body plan diversification, and further build support for an ancestral role of this pathway in early evolution of the bilaterian A/P axis. We find strong support for a role of cWnt in suppression of anterior fates and promotion of mid-axial fates, but we find no evidence that cWnt signaling plays a role in the early specification of the most posterior axial fates in S. kowalevskii. This posterior autonomy may be a conserved feature of early deuterostome axis specification

Summary of Wnts, Wnt antagonists, and Fzs receptor expression.

<p>Wnts are expressed in nested domains posteriorly, whereas <i>sfrps</i> are expressed in the anteriormost ectoderm at the blastula and gastrula stages. <i>dkk1/2/4</i> is expressed in three discrete domains of gastrula ectoderm. At juvenile stages, Sfrps are expressed in the very anterior ectoderm (apical tuft), and <i>sfrp1/5</i> is also expressed in the entire proboscis mesoderm, whereas <i>dkk1/2/4</i> is broadly expressed in the anterior ectoderm. Wnts are expressed in three discrete ectodermal domains: the base of the proboscis, the anterior trunk (over the first gill slit), and the posterior-most ectoderm. In addition, <i>wnt9</i> and <i>wntA</i> are expressed in posterior internal tissues. Fz genes are expressed in nested domains along the ectoderm. Territories are color coded: endomesoderm (grey), posterior ectoderm (dark blue), intermediate ectoderm (medium blue) and anterior ectoderm (light blue). Fz, frizzled; Sfrp, secreted frizzled-related protein.</p

Activation of the cWnt pathway leads to anterior truncation.

<p>(A-D), Treatment of embryos with the GSK3β inhibitor 1-azakenpaullone leads to a loss of proboscis at 5 μM (Aii, Bii, Cii, Dii) and to a loss of both proboscis and collar at 10 μM (Aiii, Biii, Ciii, Diii). DMSO-treated control embryos (Ai, Bi, Ci, Di). In situ hybridization for ectodermal markers of the anterior collar <i>barH</i> (A), anterior trunk <i>engrailed</i> (B), trunk <i>msx</i> (C), and posterior trunk <i>hox9/10</i> (D). Embryos at two and a half (C-D) and five (A-B) days of development. Earlier sampling at gastrula stage shows no morphological change but significant transformation of markers <i>sfrp1/5</i> (Eii) and <i>otx</i> (Fii), but no change in <i>hox9/10</i> (Gii) at 10 μM 1-azakenpaullone. DMSO control embryos (Ei, Fi, Gi). Anterior to the top, ventral to the left. (H-I), Overexpression of <i>Wnt3</i> by mRNA injection produces virtually identical phenotypes: loss of proboscis (Hii and Iii) or loss of proboscis and collar (Hiii and Iiii), depending on the strength of the phenotype. In situ hybridization for ectodermal markers of the anterior trunk <i>engrailed</i> (H) and posterior trunk <i>hox11/13c</i> (I) at three days of development (numbers indicate embryos with the displayed phenotyped over the number of analyzed embryos). Anterior to the top left, ventral to bottom left. C, control embryo; cWnt, canonical Wnt; DMSO, dimethyl sulfoxide; GSK3β, glycogen synthase kinase 3 beta.</p

RT-PCR analysis of <i>Wnt</i> and <i>Fz</i> genes expression during early development.

<p>Embryos were harvested at six different stages: oocytes, 16- to 32-cell cleavage stage embryos, late blastula, mid-gastrula, at 48 hpf, and 72 hpf. The first panel shows levels of all 13 <i>Wnt</i> genes; the second panel shows the positive control <i>actin</i> and a negative control. The third panel shows the levels of the four <i>Fz</i> receptor genes. The transcript amounts are comparable across all three panels. <i>Fz</i>, frizzled; hpf, h postfertilization; RT-PCR, reverse transcription PCR.</p