A meta-analysis of the stony coral tissue loss disease microbiome finds key bacteria in unaffected and lesion tissue in diseased colonies.

Stony coral tissue loss disease (SCTLD) has been causing significant whole colony mortality on reefs in Florida and the Caribbean. The cause of SCTLD remains unknown, with the limited concurrence of SCTLD-associated bacteria among studies. We conducted a meta-analysis of 16S ribosomal RNA gene datasets generated by 16 field and laboratory SCTLD studies to find consistent bacteria associated with SCTLD across disease zones (vulnerable, endemic, and epidemic), coral species, coral compartments (mucus, tissue, and skeleton), and colony health states (apparently healthy colony tissue (AH), and unaffected (DU) and lesion (DL) tissue from diseased colonies). We also evaluated bacteria in seawater and sediment, which may be sources of SCTLD transmission. Although AH colonies in endemic and epidemic zones harbor bacteria associated with SCTLD lesions, and aquaria and field samples had distinct microbial compositions, there were still clear differences in the microbial composition among AH, DU, and DL in the combined dataset. Alpha-diversity between AH and DL was not different; however, DU showed increased alpha-diversity compared to AH, indicating that, prior to lesion formation, corals may undergo a disturbance to the microbiome. This disturbance may be driven by Flavobacteriales, which were especially enriched in DU. In DL, Rhodobacterales and Peptostreptococcales-Tissierellales were prominent in structuring microbial interactions. We also predict an enrichment of an alpha-toxin in DL samples which is typically found in Clostridia. We provide a consensus of SCTLD-associated bacteria prior to and during lesion formation and identify how these taxa vary across studies, coral species, coral compartments, seawater, and sediment.

Freshwater unionid mussels threatened by predation of Round Goby (Neogobius melanostomus).

Indigenous freshwater mussels (Unionidae) are integral to riverine ecosystems, playing a pivotal role in aquatic food webs and providing ecological services. With populations on the decline worldwide, freshwater mussels are of conservation concern. In this study, we explore the propensity of the invasive Round Goby (Neogobius melanostomus) fish to prey upon indigenous freshwater mussels. First, we conducted lab experiments where Round Gobies were given the opportunity to feed on juvenile unionid mussels and macroinvertebrates, revealing rates and preferences of consumption. Several Round Gobies consumed whole freshwater mussels during these experiments, as confirmed by mussel counts and x-ray images of the fishes. Next, we investigated Round Gobies collected from stream habitats of the French Creek watershed, which is renowned for its unique and rich aquatic biodiversity. We developed a novel DNA metabarcoding method to identify the specific species of mussels consumed by Round Goby and provide a new database of DNA gene sequences for 25 indigenous unionid mussel species. Several of the fishes sampled had consumed indigenous mussels, including the Elktoe (non-endangered), Creeper (non-endangered), Long Solid (state endangered), and Rayed Bean (federally endangered) species. The invasive Round Goby poses a growing threat to unionid mussels, including species of conservation concern. The introduction of the invasive Round Goby to freshwaters of North America is shaping ecosystem transitions within the aquatic critical zone having widespread implications for conservation and management.

Rapid Implementation of High-Frequency Wastewater Surveillance of SARS-CoV-2.

There have been over 507 million cases of COVID-19, the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in 6 million deaths globally. Wastewater surveillance has emerged as a valuable tool in understanding SARS-CoV-2 burden in communities. The National Wastewater Surveillance System (NWSS) partnered with the United States Geological Survey (USGS) to implement a high-frequency sampling program. This report describes basic surveillance and sampling statistics as well as a comparison of SARS-CoV-2 trends between high-frequency sampling 3-5 times per week, referred to as USGS samples, and routine sampling 1-2 times per week, referred to as NWSS samples. USGS samples provided a more nuanced impression of the changes in wastewater trends, which could be important in emergency response situations. Despite the rapid implementation time frame, USGS samples had similar data quality and testing turnaround times as NWSS samples. Ensuring there is a reliable sample collection and testing plan before an emergency arises will aid in the rapid implementation of a high-frequency sampling approach. High-frequency sampling requires a constant flow of information and supplies throughout sample collection, testing, analysis, and data sharing. High-frequency sampling may be a useful approach for increased resolution of disease trends in emergency response.

Novel microbiome dominated by Arcobacter during anoxic excurrent flow from an ocean blue hole in Andros Island, The Bahamas.

Andros Island, The Bahamas, composed of porous carbonate rock, has about 175 inland blue holes and over 50 known submerged ocean caves along its eastern barrier reef. These ocean blue holes can have both vertical and horizontal zones that penetrate under the island. Tidal forces drive water flow in and out of these caves. King Kong Cavern has a vertical collapse zone and a deep penetration under Andros Island that emits sulfidic, anoxic water and masses of thin, mucoid filaments ranging to meters in length and off-white turbid water during ebb flow. Our objective was to determine the microbial composition of this mucoid material and the unconsolidated water column turbidity based on the concept that they represent unique lithoautotrophic microbial material swept from the cave into the surrounding ocean. Bacterial DNA extracted from these filaments and surrounding turbid water was characterized using PCR that targeted a portion of the 16S rRNA gene. The genus Arcobacter dominated both the filaments and the water column above the cave entrance. Arcobacter nitrofigilis and Arcobacter sp. UDC415 in the mucoid filaments accounted for as much as 80% of mapped DNA reads. In the water column Arcobacter comprised from 65% to over 85% of the reads in the depth region from about 18 m to 34 m. Bacterial species diversity was much higher in surface water and in water deeper than 36 m than in the intermediate zone. Community composition indicates that ebb flow from the cavern influences the entire water column at least to within 6 m of the surface and perhaps the near surface as well.

Capture of Environmental DNA (eDNA) from Water Samples by Flocculation.

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species including invasive and (or) species at risk is the goal, typically accomplished by testing water and sediment for the presence of characteristic DNA signatures. Reliable and efficient procedures for the capture of eDNA are required, especially those that can be performed easily in the field by personnel with limited training and citizen scientists. The capture of eDNA using membrane filtration is widely used currently. This approach has inherent issues that include the choice of filter material and porosity, filter fouling, and time required on site for the process to be performed. Flocculation offers an alternative that can be easily implemented and applied to sampling regimes that strive to cover broad territories in limited time.

The complete maternal mitochondrial genome sequences of two imperiled North American freshwater mussels: Alasmidonta heterodon and Alasmidonta varicosa (Bivalvia: Unionoida: Unionidae).

The freshwater mussels Alasmidonta heterodon and A. varicosa historically inhabited rivers along the North American Atlantic coast from the Carolinas, U.S.A., to New Brunswick, CA. However, many populations have been extirpated, and A. heterodon is now federally listed in the U.S.A. as endangered, and both A. heterodon and A. varicosa are listed as vulnerable on the IUCN Red List. To facilitate genetic study of these species, we sequenced the complete female mitochondrial genomes of A. heterodon (15,909 bp; GenBank accession no. MG905826), and A. varicosa (15,693 bp; GenBank accession no. MG938673). Both mitogenomes contained 14 protein coding genes, 2 rRNA genes, and 22 tRNAs with the same gene order as reported for other members of the subfamily Anodontinae. When these two genomes were put into a phylogenetic context with other members of the Unionidae, they clustered together with other species in the subfamily Anodontinae, Tribe Anodontini.

An investigation of the bactericidal activity of selected essential oils to Aeromonas spp.

Diseases of fishes caused by Aeromonas spp. are common, have broad host ranges and may cause high mortality. Treatments of captive-reared populations using antimicrobials are limited with concerns for bacterial resistance development and environmental dissemination. This study was done to determine whether selected plant-derived essential oils were bactericidal to Aeromonas spp. Initially, twelve essential oils were evaluated using a disk diffusion assay to an isolate of A. salmonicida subsp. salmonicida, cause of fish furunculosis. The greatest zones of inhibition were obtained with oils of cinnamon Cinnamomum cassia, oregano Origanum vulgare, lemongrass Cymbopogon citratus and thyme Thymus vulgaris. Minimum bactericidal concentrations (MBC's) were determined for these four oils, Allimed® (garlic extract, Allium sativum) and colloidal silver to sixty-nine isolates representing nine Aeromonas spp. The lowest mean MBCs (0.02-0.04%) were obtained with three different sources of cinnamon oil. MBCs for three sources of oregano and lemongrass oils ranged from 0.14% to 0.30% and 0.10% to 0.65%, respectively, and for two thyme oils were 2.11% and 2.22%. The highest concentration (5%) of Allimed® tested resulted in MBCs to twelve isolates. A concentration of silver greater than 15 mg/L would be required to determine MBCs for all but one isolate.

Real-time PCR detection and quantification of nine potential sources of fecal contamination by analysis of mitochondrial cytochrome b targets.

We designed and tested real-time PCR probe/primer sets to detect and quantify Cytochrome b sequences of mitochondrial DNA (mtDNA) from nine vertebrate species of pet (dog), farm (cow, chicken, sheep, horse, pig), wildlife (Canada goose, white-tailed deer), and human. Linear ranges of the assays were from 10(1) to 10(8) copies/microl. To formally test the performance of the assays, twenty blinded fecal suspension samples were analyzed by real-time PCR to identify the source of the feces. Sixteen of the twenty samples were correctly and unambiguously identified. Average sensitivity was calculated to be 0.850, while average specificity was found to be 0.994. One beef cow sample was not detected, but mtDNA from 11 other beef cattle of both sexes and varying physiological states was found in concentrations similar (3.45 x 10(7) copies/g) to thatfound in human feces (1.1 x 10(7) copies/g). Thus, environmental conditions and sample handling are probably important factors for successful detection of fecal mtDNA. When sewage samples were analyzed, only human mtDNA (7.2 x 10(4) copies/ 100 mL) was detected. With a detection threshold of 250 copies/reaction, an efficient concentration and purification method resulted in a final detection limit for human feces of 1.8 mg/ 100 mL water.

Morphology and 18S rDNA of Henneguya gurlei (Myxosporea) from Ameiurus nebulosus (Siluriformes) in North Carolina.

Henneguya gurlei was isolated from Ameiurus nebulosus captured in North Carolina and redescribed using critical morphological features and 18S small-subunit ribosomal RNA (SSU rDNA) gene sequence. Plasmodia are white, spherical, or subspherical, occur in clusters, measure up to 1.8 mm in length, and are located on the dorsal, pectoral, and anal fins. Histologically, plasmodia are located in the dermis and subdermally, and the larger cysts disrupt the melanocyte pigment layer. The spore body is lanceolate, 18.2 +/- 0.3 microm (range 15.7-20.3) in length, and 5.4 +/- 0.1 microm (range 3.8-6.1) in width in valvular view. The caudal appendages are 41.1 +/- 1.1 microm (range 34.0-49.7) in length. Polar capsules are pyriform and of unequal size. The longer polar capsule measures 6.2 +/- 0.1 microm (range 5.48-7.06), while the shorter is 5.7 +/- 0.1 microm (range 4.8-6.4) in length. Polar capsule width is 1.2 +/- 0.03 microm (range 1.0-1.54). The total length of the spore is 60.9 +/- 1.2 microm (range 48.7-68.5). Morphologically, this species is similar to other species of Henneguya that are known to infect ictalurids. Based on SSU rDNA sequences, this species is most closely related to H. exilis and H. ictaluri, which infect Ictalurus punctatus.

The proinflammatory cytokine interleukin-18 alters multiple signaling pathways to inhibit natural killer cell death.

The proinflammatory cytokine, interleukin-18 (IL-18), is a natural killer (NK) cell activator that induces NK cell cytotoxicity and interferon-gamma (IFN-gamma) expression. In this report, we define a novel role for IL-18 as an NK cell protective agent. Specifically, IL-18 prevents NK cell death initiated by different and distinct stress mechanisms. IL-18 reduces NK cell self-destruction during NK-targeted cell killing, and in the presence of staurosporin, a potent apoptotic inducer, IL-18 reduces caspase-3 activity. The critical regulatory step in this process is downstream of the mitochondrion and involves reduced cleavage and activation of caspase-9 and caspase-3. The ability of IL-18 to regulate cell survival is not limited to a caspase death pathway in that IL-18 augments tumor necrosis factor (TNF) signaling, resulting in increased and prolonged mRNA expression of c-apoptosis inhibitor 2 (cIAP2), a prosurvival factor and caspase-3 inhibitor, and TNF receptor-associated factor 1 (TRAF1), a prosurvival protein. The cumulative effects of IL-18 define a novel role for this cytokine as a molecular survival switch that functions to both decrease cell death through inhibition of the mitochondrial apoptotic pathway and enhance TNF induction of prosurvival factors.

Effects of water temperature and substrate type on spore production and release in eastern Tubifex tubifex worms infected with Myxobolus cerebralis.

Eastern Tubifex tubifex worms were exposed to Myxobolus cerebralis spores at 9, 13, 17, and 20 C in 1-L jars that contained sand, mud, or leaf litter as substrata. Beginning 60 days after exposure, water from each jar was filtered daily and examined for the presence of waterborne triactinomyxon spores (TAMs). On discovering a single TAM from an experimental jar, 48 T. tubifex worms from that jar were placed individually into 24-well plates. Spores released from individual infected T. tubifex worms were quantified to determine the first day of TAM release from infected worms, the infection rate, the total number of TAMs released per worm, and the duration of release. No TAMs were found in any of the jars incubated at 20 C or in uninfected, control worms at any temperature. The total number of TAMs released by infected worms in mud and sand was highest at 13 C compared with other temperatures. Infection rates among individual worms increased with temperature between 9 and 17 C. Higher temperatures (up to 17 C) induced earlier TAM releases among infected worms, and substratum did not influence this production parameter. The average duration of TAM release decreased as the temperature increased from 9 to 17 C, and there was a significant effect of substratum in the groups maintained at 13 and 17 C. In all temperature treatments between 9 and 17 C, the duration of release was least in the worms maintained in leaf litter, as was the total number of TAMs released during the experimental period and the median number of TAMs per production day.

IL-2 and IL-12 alter NK cell responsiveness to IFN-gamma-inducible protein 10 by down-regulating CXCR3 expression.

Cytokine treatment of NK cells results in alterations in multiple cellular responses that include cytotoxicity, cytokine production, proliferation, and chemotaxis. To understand the molecular mechanisms underlying these responses, microarray analysis was performed and the resulting gene expression patterns were compared between unstimulated, IL-2, IL-2 plus IL-12, and IL-2 plus IL-18-stimulated NK92 cells. RNase protection assays and RT-PCR confirmed microarray predictions for changes in mRNA expression for nine genes involved in cell cycle progression, signal transduction, transcriptional activation, and chemotaxis. Multiprobe RNase protection assay also detected changes in the expression of CCR2 mRNA, a gene that was not imprinted on the microarray. We subsequently expanded our search for other chemokine receptor genes absent from the microarray and found an IL-2- and IL-12-dependent decrease in CXCR3 receptor mRNA expression in NK92 cells. A detailed analysis of CXCR3 expression in primary NK cells revealed that an IL-2 and an IL-12 together significantly decreased the CXCR3 receptor mRNA and receptor surface expression by 6 and 24 h of treatment, respectively. This decrease in receptor expression was associated with a significant reduction in chemotaxis in the presence of IFN-gamma-inducible protein-10. The decline in CXCR3 mRNA was due to transcriptional and posttranscriptional mechanisms as the addition of actinomycin D to IL-2- and IL-12-treated NK92 slightly altered the half-life of the CXCR3 mRNA. Collectively, these data suggest that IL-2 and IL-12 directly affect NK cell migratory ability by rapid and direct down-regulation of chemokine receptor mRNA expression.