Biochemical and Ultrastructural Evidence for the Origin of Whitings: A Biologically Induced Calcium Carbonate Precipitation Mechanism

We propose that Bahamian whitings, floating patches of lime mud, are in part the result of biological precipitation of calcium carbonate induced by picoplankton and cellular components. The cells may act as nucleation sites for crystallization from seawater supersaturated with CaCO3. Our model represents a new hypothesis concerning Bahamian whiting formation and is based on several lines of evidence. Biochemical data suggest that the macromolecules from carbonate suspended in Bahama Banks whitings are distinct from those found in the lime mud sediment producer Penicillus and from bottom sediment. Direct ultrastructural evidence indicates that mineralization occurs on the surfaces of picoplankton cells and degrading organic cellular components. The organic constituents include, but are not restricted to, whole and fragmented algal cells and composite structures ∼20-30 μm in diameter. The observed calcium carbonate crystals are distinct from skeletal debris. Because of the magnitude of the whiting phenomenon, calculations of carbonate sediment production budgets should include the potential contribution of calcium carbonate produced by epicefular precipitation. Knowledge of carbonate mud genesis is critical to interpretation of ancient occurrences of lime mud as well as to understanding ancient and global carbon cycles

Coral Ultrastructural Response to Elevated pCO2 and Nutrients During Tissue Repair and Regeneration

Corals and coral reefs have recently experienced widespread decline attributed to anthropogenic pressure on reef systems. Studies have demonstrated that nutrient and pCO2 stress effect coral growth and calcification, but study of specific effects on coral tissue is lacking. The objective of this research was to examine wound healing in corals and how it is affected by exposure to elevated nutrients and pCO2. Coral tissue repair and regeneration during wound healing in Montastraea cavernosa and Porites astreoides were assessed histologically and ultrastructurally by examining colony fragments exposed to elevated nitrate, phosphate, and pCO2. In M. cavernosa, tissue repair was facilitated by granular amoebocytes, and the zooxanthellae population size increased under enriched nutrient conditions. In P. astreoides, zooxanthellae chloroplasts were markedly abnormal in phosphate-enriched corals, and the concentration of chromophore cells at the healing tissue front was markedly lower under elevated nutrient conditions. The area of wound healed was higher after 14 days under every experimental condition in M. cavernosa compared to P. astreoides. In both species, phosphate enrichment had the most deleterious effect on repair and regeneration

Coccolith Morphology and Paleoclimatology - 2. Cell Ultrastructure and Formation of Coccoliths in Cyclococcolithina leptopora (Murray and Blackman) Wilcoxon and Gephyrocapsa oceanica Kamptner.

Current interest in utilization of coccoliths for paleoclimate reconstruction necessitates background information on environmental limits for growth and coccolith production as well as examination of cell ultrastructure in specimens collected in the field and in cultured representatives. Successful isolation of the two geologically important species Gephyrocapsa oceanica (strain A674) and Cyclococcolithina leptopora (strain A650) allows investigation of ultrastructure in cultured forms. Fine structure of cells and coccoliths was observed in the SEM using critical point dried preparations and ultrastructure was examined with the transmission electron microscope. Coccoliths are formed intracellularly and appear to form within Golgi-derived vesicles located near the nuclear membrane. Formation and development of coccoliths in the two species resemble these processes in Emiliania huxleyi but differ from those of Cricosphaera carterae, notably in the absence of coccolithosomes and scales and in the fact that coccoliths are produced intracellularly one at a time

Insights into Migration and Development of Coral Black Band Disease Based on Fine Structure Analysis

In many diverse ecosystems, ranging from natural surfaces in aquatic ecosystems to the mammalian gut and medical implants, bacterial populations and communities exist as biofilms. While the process of biofilm development has been well-studied for those produced by unicellular bacteria such Pseudomonas aeruginosa, little is known about biofilm development associated with filamentous microorganisms. Black band disease (BBD) of corals is characterized as a polymicrobial biofilm (mat) community, visually-dominated by filamentous cyanobacteria. The mat migrates across a living coral host, completely lysing coral tissue and leaving behind exposed coral skeleton. It is the only known cyanobacterial biofilm that migrates across a substratum, thus eliciting questions about the mechanisms and unique characteristics of this system. Fragments of the coral Montastraea annularis, five artificially infected with BBD and two collected from a naturally BBD-infected colony, were used to address these questions by detailed examination using scanning and transmission electron microscopy (SEM and TEM). In areas close to the interface of coral tissue and the mature disease band two types of clusters of cyanobacteria were observed, one with random orientation and one with parallel orientation of filaments. The latter exhibited active secretion of extracellular polysaccharide (EPS) while the randomly oriented clusters did not. Within the well developed band cyanobacterial filaments were observed to be embedded in EPS and were present as layers of filaments in parallel orientation. These observations suggest that BBD cyanobacteria orient themselves and produce EPS in a sequential process during migration to form the complex BBD matrix

Ultrastructural and Histological Analysis of Dark Spot Syndrome in Siderastrea siderea and Agaricia agaricites

Dark Spot Syndrome (DSS) typically manifests in scleractinian corals as lesions of varying color, size, shape and location that can result in skeletal changes and tissue death. A causative agent for DSS has not yet been identified. The objective of this study was histological and ultrastructural comparison of the cellular and skeletal characteristics of DSS-affected and healthy Siderastrea siderea and Agaricia agaricites. The greater resolution possible with transmission electron microscopy (TEM) revealed microbial activity and tissue changes not resolvable utilizing histology. DSS-affected tissue had less integrity, with increasing cellular degradation and vacuolization. A high concentration of electron dense inclusions, which appear to be zymogen granules, was concentrated in the calicodermis and adjacent gastrodermal layer. Numerous endolithic fungal cells were observed directly adjacent to the calicodermis in DSS-affected S. siderea. Numerous unidentified endolithic cells were observed directly adjacent to the calicodermis in DSS-affected A. agaricites. These observations suggest that the coral may be using a digestive enzyme as a defensive mechanism against endolithic cellular invasion

Microscopic and Genetic Characterization of Bacterial Symbionts With Bioluminescent Potential in Pyrosoma atlanticum

The pelagic tunicate pyrosome, Pyrosoma atlanticum, is known for its brilliant bioluminescence, but the mechanism causing this bioluminescence has not been fully characterized. This study identifies the bacterial bioluminescent symbionts of P. atlanticum collected in the northern Gulf of Mexico using several methods such as light and electron microscopy, as well as molecular genetics. The bacteria are localized within the pyrosome light organs. Greater than 50% of the bacterial taxa present in the tunicate samples were the bioluminescent symbiotic bacteria Vibrionaceae as determined by utilizing current molecular genetics methodologies. A total of 396K MiSeq16S rRNA reads provided total pyrosome microbiome profiles to determine bacterial symbiont taxonomy. After comparing with the Silva rRNA database, a Photobacterium sp. r33-like bacterium (which we refer to as “Photobacterium Pa-1”) matched at 99% sequence identity as the most abundant bacteria within Pyrosoma atlanticum samples. Specifically designed 16S rRNA V4 probes for fluorescence in situ hybridization (FISH) verified the Photobacterium Pa-1 location as internally concentrated along the periphery of each dual pyrosome luminous organ. While searching for bacterial lux genes in two tunicate samples, we also serendipitously generated a draft tunicate mitochondrial genome that can be used for Pyrosoma atlanticum identification. Scanning (SEM) and transmission (TEM) electron microscopy confirmed the presence of intracellular rod-like bacteria in the light organs. This intracellular localization of bacteria may represent bacteriocyte formation reminiscent of other invertebrates

Characterization of the microbiome and bioluminescent symbionts across life stages of ceratioid anglerfishes of the gulf of mexico

The interdependence of diverse organisms through symbiosis reaches even the deepest parts of the oceans. As part of the DEEPEND project (deependconsortium.org) research on deep Gulf of Mexico biodiversity, we profiled the bacterial communities (‘microbiomes’) and luminous symbionts of 36 specimens of adult and larval deep-sea anglerfishes of the suborder Ceratioidei using 16S rDNA. Transmission Electron Microscopy was used to characterize the location of symbionts in adult light organs (esca). Whole larval microbiomes, and adult skin and gut microbiomes, were dominated by bacteria in the genera Moritella and Pseudoalteromonas genera. 16S rDNA sequencing results from adult fishes corroborate the previously published identity of ceratioid bioluminescent symbionts and support findings that these symbionts do not consistently exhibit host specificity at the host family level. Bioluminescent symbiont amplicon sequence variants (ASVs) were absent from larval ceratioid samples, but were found at all depths in the seawater, with a highest abundance found at mesopelagic depths. As adults spend the majority of their lives in the meso and bathypelagic, the trend in symbiont abundance is consistent with their life history. These findings support the hypothesis that bioluminescent symbionts are not present throughout host development, and that ceratioids acquire their bioluminescent symbionts from the environment

Two distinct microbial communities revealed in the sponge Cinachyrella

Marine sponges are vital components of benthic and coral reef ecosystems, providing shelter and nutrition for many organisms. In addition, sponges act as an essential carbon and nutrient link between the pelagic and benthic environment by filtering large quantities of seawater. Many sponge species harbor a diverse microbial community (including Archaea, Bacteria and Eukaryotes), which can constitute up to 50% of the sponge biomass. Sponges of the genus Cinachyrella are common in Caribbean and Floridian reefs and their archaeal and bacterial microbiomes were explored here using 16S rRNA gene tag pyrosequencing. Cinachyrella specimens and seawater samples were collected from the same South Florida reef at two different times of year. In total, 639 OTUs (12 archaeal and 627 bacterial) belonging to 2 archaeal and 21 bacterial phyla were detected in the sponges. Based on their microbiomes, the six sponge samples formed two distinct groups, namely sponge group 1 (SG1) with lower diversity (Shannon-Wiener index: 3.73 ± 0.22) and SG2 with higher diversity (Shannon-Wiener index: 5.95 ± 0.25). Hosts’ 28S rRNA gene sequences further confirmed that the sponge specimens were composed of two taxa closely related to Cinachyrella kuekenthalli. Both sponge groups were dominated by Proteobacteria, but Alphaproteobacteria were significantly more abundant in SG1. SG2 harbored many bacterial phyla (>1% of sequences) present in low abundance or below detection limits (<0.07%) in SG1 including: Acidobacteria, Chloroflexi, Gemmatimonadetes, Nitrospirae, PAUC34f, Poribacteria, and Verrucomicrobia. Furthermore, SG1 and SG2 only had 95 OTUs in common, representing 30.5 and 22.4% of SG1 and SG2’s total OTUs, respectively. These results suggest that the sponge host may exert a pivotal influence on the nature and structure of the microbial community and may only be marginally affected by external environment parameters.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by the Frontiers Research Foundation. The published article can be found at: http://www.frontiersin.org/Microbiology.Keywords: diversity, 16S rRNA, marine sponge, microbiome, pyrosequencing, archaea, symbiont

Netosis and Inflammasomes in Large Vessel Occlusion Thrombi

The inflammatory response appears to play a critical role in clotting in which neutrophil extracellular traps (NETs) are the major drivers of thrombosis in acute ischemic stroke (AIS). The inflammasome is an innate immune complex involved in the activation of interleukin (IL)-18 and IL-1β through caspase-1, but whether the inflammasome plays a role in NETosis in AIS remains poorly understood. Here we assessed the levels of inflammasome signaling proteins in NETs and their association with clinical and procedural outcomes of mechanical thrombectomy for AIS. Electron microscopy and immunofluorescence indicate the presence of NETs in thrombi of patients with AIS. Moreover, the inflammasome signaling proteins caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) were also present in clots associated with the marker of NETosis citrullinated histone H (CitH3). Analysis of protein levels by a simple plex assay show that caspase-1, ASC and interleukin (IL)-1β were significantly elevated in clots when compared to plasma of AIS patients and healthy controls, while IL-18 levels were lower. Moreover, multivariate analyses show that IL-1β levels in clots contribute to the number of passes to achieve complete recanalization, and that ASC, caspase-1 and IL-18 are significant contributors to time to recanalization. Thus, inflammasome proteins are elevated in NETs present in thrombi of patients with AIS that contribute to poor outcomes following stroke.

Ensuring Quality in AFRINEST and SATT: Clinical Standardization and Monitoring

Background: Three randomized open-label clinical trials [Simplified Antibiotic Therapy Trial (SATT) Bangladesh, SATT Pakistan and African Neonatal Sepsis Trial (AFRINEST)] were developed to test the equivalence of simplified antibiotic regimens compared with the standard regimen of 7 days of parenteral antibiotics. These trials were originally conceived and designed separately; subsequently, significant efforts were made to develop and implement a common protocol and approach. Previous articles in this supplement briefly describe the specific quality control methods used in the individual trials; this article presents additional information about the systematic approaches used to minimize threats to validity and ensure quality across the trials. Methods: A critical component of quality control for AFRINEST and SATT was striving to eliminate variation in clinical assessments and decisions regarding eligibility, enrollment and treatment outcomes. Ensuring appropriate and consistent clinical judgment was accomplished through standardized approaches applied across the trials, including training, assessment of clinical skills and refresher training. Standardized monitoring procedures were also applied across the trials, including routine (day-to-day) internal monitoring of performance and adherence to protocols, systematic external monitoring by funding agencies and external monitoring by experienced, independent trial monitors. A group of independent experts (Technical Steering Committee/Technical Advisory Group) provided regular monitoring and technical oversight for the trials. Conclusions: Harmonization of AFRINEST and SATT have helped to ensure consistency and quality of implementation, both internally and across the trials as a whole, thereby minimizing potential threats to the validity of the trials’ results