Bioelectric-calcineurin signaling module regulates allometric growth and size of the zebrafish fin

AbstractThe establishment of relative size of organs and structures is paramount for attaining final form and function of an organism. Importantly, variation in the proportions of structures frequently underlies adaptive change in morphology in evolution and maybe a common mechanism underlying selection. However, the mechanism by which growth is integrated within tissues during development to achieve proper proportionality is poorly understood. We have shown that signaling by potassium channels mediates coordinated size regulation in zebrafish fins. Recently, calcineurin inhibitors were shown to elicit changes in zebrafish fin allometry as well. Here, we identify the potassium channelkcnk5bas a key player in integrating calcineurin’s growth effects, in part through regulation of the cytoplasmic C-terminus of the channel. We propose that the interaction between Kcnk5b and calcineurin acts as a signaling node to regulate allometric growth. Importantly, we find that this regulation is epistatic to inherent mechanisms instructing overall size as inhibition of calcineurin is able to bypass genetic instruction of size as seen insofand wild-type fins, however, it is not sufficient to re-specify positional memory of size of the fin. These findings integrate classic signaling mediators such as calcineurin with ion channel function in the regulation of size and proportion during growth.</jats:p

No evidence of transmission of grapevine leafroll-associated viruses by phylloxera (Daktulosphaira vitifoliae).

Grapevine leafroll disease is associated with several species of phloem-limited grapevine leafrollassociated viruses (GLRaV), some of which are transmitted by mealybugs and scale insects. The grape phylloxera, Daktulosphaira vitifoliae (Fitch) Biotype A (Hemiptera: Phylloxeridae), is a common vineyard pest that feeds on the phloem of vine roots. There is concern that these insects may transmit one or more GLRaV species, particularly GLRaV-2, a species in the genus Closterovirus. A field survey was performed in vineyards with a high incidence of grapevine leafroll disease and D. vitifoliae was assessed for acquisition of GLRaV. In greenhouse experiments, the ability of D. vitifoliae to transmit GLRaV from infected root sections or vines to co-planted virus-free recipient vines was tested. There were no GLRaV-positive D. vitifoliae in the field survey, nor did D. vitifoliae transmit GLRaV- 1, ?2, ?3, or -4LV in greenhouse transmission experiments. Some insects tested positive for GLRaV after feeding on infected source vines in the greenhouse, however there was no evidence of virus transmission to healthy plants. These findings, in combination with the sedentary behaviour of the soil biotype of D. vitifoliae, make it unlikely that D. vitifoliae is a vector of any GLRaV.DOI: 10.1007/s10658-016-1049-

Bioelectric Signaling Regulates Size in Zebrafish Fins

The scaling relationship between the size of an appendage or organ and that of the body as a whole is tightly regulated during animal development. If a structure grows at a different rate than the rest of the body, this process is termed allometric growth. The zebrafish another longfin (alf) mutant shows allometric growth resulting in proportionally enlarged fins and barbels. We took advantage of this mutant to study the regulation of size in vertebrates. Here, we show that alf mutants carry gain-of-function mutations in kcnk5b, a gene encoding a two-pore domain potassium (K+) channel. Electrophysiological analysis in Xenopus oocytes reveals that these mutations cause an increase in K+ conductance of the channel and lead to hyperpolarization of the cell. Further, somatic transgenesis experiments indicate that kcnk5b acts locally within the mesenchyme of fins and barbels to specify appendage size. Finally, we show that the channel requires the ability to conduct K+ ions to increase the size of these structures. Our results provide evidence for a role of bioelectric signaling through K+ channels in the regulation of allometric scaling and coordination of growth in the zebrafish

Occurrence of Grapevine Leafroll-Associated Virus Complex in Napa Valley

Grapevine leafroll disease (GLD) is caused by a complex of several virus species (grapevine leafroll-associated viruses, GLRaV) in the family Closteroviridae. Because of its increasing importance, it is critical to determine which species of GLRaV is predominant in each region where this disease is occurring. A structured sampling design, utilizing a combination of RT-PCR based testing and sequencing methods, was used to survey GLRaVs in Napa Valley (California, USA) vineyards (n = 36). Of the 216 samples tested for GLRaV-1, -2, -3, -4, -5, and -9, 62% (n = 134) were GLRaV positive. Of the positives, 81% (n = 109) were single infections with GLRaV-3, followed by GLRaV-2 (4%, n = 5), while the remaining samples (15%, n = 20) were mixed infections of GLRaV-3 with GLRaV-1, 2, 4, or 9. Additionally, 468 samples were tested for genetic variants of GLRaV-3, and of the 65% (n = 306) of samples positive for GLRaV-3, 22% were infected with multiple GLRaV-3 variants. Phylogenetic analysis utilizing sequence data from the single infection GLRaV-3 samples produced seven well-supported GLRaV-3 variants, of which three represented 71% of all GLRaV-3 positive samples in Napa Valley. Furthermore, two novel variants, which grouped with a divergent isolate from New Zealand (NZ-1), were identified, and these variants comprised 6% of all positive GLRaV-3 samples. Spatial analyses showed that GLRaV-3a, 3b, and 3c were not homogeneously distributed across Napa Valley. Overall, 86% of all blocks (n = 31) were positive for GLRaVs and 90% of positive blocks (n = 28) had two or more GLRaV-3 variants, suggesting complex disease dynamics that might include multiple insect-mediated introduction events

Development of a Multiplex PCR for Identification of Vineyard Mealybugs

A simple molecular tool was developed and tested to identify seven mealybug species found in North American vineyards: Pseudococcus maritimus Ehrhorn, Pseudococcus viburni (Signoret), Pseudococcus longispinus (Targioni-Tozzeti), Pseudococcus calceolariae (Maskell), Planococcus fleas (Signoret), Planococcus citri (Risso), and Ferrisia gilli Gullan. The developed multiplex PCR is based on the mitochondrial cytochrome c oxidase subunit one gene. In tests, this single-step multiplex PCR correctly identified 95 of 95 mealybug samples, representing all seven species and collected from diverse geographic regions. To test the sensitivity, single specimen samples with different Pl. ficus developmental stages (egg to adult female and adult male) were processed PCR and the resulting output provided consistent positive identification. To test the utility of this protocol for adult males caught in sex baited pheromone traps, Pl. ficus adult males were placed in pheromone traps, aged at a constant temperature of 26 +/- 2 degrees C, and processed with the multiplex each day thereafter for 8 d. Results showed consistent positive identification for up to 6 d (range, 6-8 d). Results are discussed with respect to the usefulness of this molecular tool for the identification of mealybugs in pest management programs and biosecurity of invasive mealybugs.Keywords: Pest Identification, Vineyards, Multiplex PCR, Pseudococcus, Planococcu

High-throughput isolation of circulating tumor DNA

The emerging interest in circulating tumor DNA (ctDNA) analyses for clinical trials has necessitated the development of a high-throughput method for fast, reproducible, and efficient isolation of ctDNA. Currently, the majority of ctDNA studies use the manual QIAamp (QA) platform to isolate DNA from blood. The purpose of this study was to compare two competing automated DNA isolation platforms [Maxwell (MX) and QIAsymphony (QS)] to the current ‘gold standard’ QA to facilitate high-throughput processing of samples in prospective trials. We obtained blood samples from healthy blood donors and metastatic cancer patients for plasma isolation. Total cell-free DNA (cfDNA) quantity was assessed by TERT quantitative PCR. Recovery efficiency was investigated by quantitative PCR analysis of spiked-in synthetic plant DNA. In addition, a b-actin fragmentation assay was performed to determine the amount of contamination by genomic DNA from lysed leukocytes. ctDNA quality was assessed by digital PCR for somatic variant detection. cfDNA quantity and recovery efficiency were lowest using the MX platform, whereas QA and QS showed a comparable performance. All platforms preferentially isolated small (136 bp) DNA fragments over large (420 and 2000 bp) DNA fragments. Detection of the number variant and wild-type molecules was most comparable between QA and QS. However, there was no significant difference in variant allele frequency comparing QS and MX to QA. In summary, we show that the QS platform has comparable performance to QA, the ‘gold standard’, and outperformed the MX platform depending on the readout used. We conclude that the QS can replace the more laborious QA platform, especially when high-throughput cfDNA isolation is needed

Species-Specific Effects of Epigeic Earthworms on Microbial Community Structure during First Stages of Decomposition of Organic Matter

Background: Epigeic earthworms are key organisms in organic matter decomposition because of the interactions they establish with microorganisms. The earthworm species and the quality and/or substrate availability are expected to be major factors influencing the outcome of these interactions. Here we tested whether and to what extent the epigeic earthworms Eisenia andrei, Eisenia fetida and Perionyx excavatus, widely used in vermicomposting, are capable of altering the microbiological properties of fresh organic matter in the short-term. We also questioned if the earthworm-induced modifications to the microbial communities are dependent on the type of substrate ingested. Methodology/Principal Findings: To address these questions we determined the microbial community structure (phospholipid fatty acid profiles) and microbial activity (basal respiration and microbial growth rates) of three types of animal manure (cow, horse and rabbit) that differed in microbial composition, after being processed by each species of earthworm for one month. No differences were found between earthworm-worked samples with regards to microbial community structure, irrespective of type of manure, which suggests the existence of a bottleneck effect of worm digestion on microbial populations of the original material consumed. Moreover, in mesocosms containing cow manure the presence of E. andrei resulted not only in a decrease in bacterial and fungal biomass, but also in a reduced bacterial growth rate and total microbial activity, while no such reduction was found with E. fetida and P. excavatus

The Secret Life of the Anthrax Agent Bacillus anthracis: Bacteriophage-Mediated Ecological Adaptations

Ecological and genetic factors that govern the occurrence and persistence of anthrax reservoirs in the environment are obscure. A central tenet, based on limited and often conflicting studies, has long held that growing or vegetative forms of Bacillus anthracis survive poorly outside the mammalian host and must sporulate to survive in the environment. Here, we present evidence of a more dynamic lifecycle, whereby interactions with bacterial viruses, or bacteriophages, elicit phenotypic alterations in B. anthracis and the emergence of infected derivatives, or lysogens, with dramatically altered survival capabilities. Using both laboratory and environmental B. anthracis strains, we show that lysogeny can block or promote sporulation depending on the phage, induce exopolysaccharide expression and biofilm formation, and enable the long-term colonization of both an artificial soil environment and the intestinal tract of the invertebrate redworm, Eisenia fetida. All of the B. anthracis lysogens existed in a pseudolysogenic-like state in both the soil and worm gut, shedding phages that could in turn infect non-lysogenic B. anthracis recipients and confer survival phenotypes in those environments. Finally, the mechanism behind several phenotypic changes was found to require phage-encoded bacterial sigma factors and the expression of at least one host-encoded protein predicted to be involved in the colonization of invertebrate intestines. The results here demonstrate that during its environmental phase, bacteriophages provide B. anthracis with alternatives to sporulation that involve the activation of soil-survival and endosymbiotic capabilities