Lithium chloride outperformed oxalic acid sublimation in a preliminary experiment for Varroa mite control in pre-wintering honey bee colonies.

Since lithium salts were demonstrated to be very effective for the potential control of Varroa destructor, a highly detrimental parasite of honey bee (Apis mellifera), no studies have been reported on their comparison with any commonly used varroicides in commercial bee colonies. In this study we compared the effectiveness of lithium chloride to that of oxalic acid, a widely used miticide. The results of the present study confirm that lithium has superior efficacy to oxalic acid sublimation both as a main or a supplementary pre-wintering treatment at moderate infestation levels, restricted to certain pre-wintering conditions. Considering its easy implementation in apicultural practice and its twofold mode of action, trickling would be the preferred way of administration after the use of lithium salts as varroicides is authorised.

The nonstop decay and the RNA silencing systems operate cooperatively in plants.

Translation-dependent mRNA quality control systems protect the protein homeostasis of eukaryotic cells by eliminating aberrant transcripts and stimulating the decay of their protein products. Although these systems are intensively studied in animals, little is known about the translation-dependent quality control systems in plants. Here, we characterize the mechanism of nonstop decay (NSD) system in Nicotiana benthamiana model plant. We show that plant NSD efficiently degrades nonstop mRNAs, which can be generated by premature polyadenylation, and stop codon-less transcripts, which are produced by endonucleolytic cleavage. We demonstrate that in plants, like in animals, Pelota, Hbs1 and SKI2 proteins are required for NSD, supporting that NSD is an ancient and conserved eukaryotic quality control system. Relevantly, we found that NSD and RNA silencing systems cooperate in plants. Plant silencing predominantly represses target mRNAs through endonucleolytic cleavage in the coding region. Here we show that NSD is required for the elimination of 5' cleavage product of mi- or siRNA-guided silencing complex when the cleavage occurs in the coding region. We also show that NSD and nonsense-mediated decay (NMD) quality control systems operate independently in plants.

Robotic Transanal Minimally Invasive Surgery for the Excision of Rectal Neoplasia: Clinical Experience With 58 Consecutive Patients.

BACKGROUND: Given the significant perioperative risks and costs of total mesorectal excision, minimally invasive transanal surgical approaches have grown in popularity for early rectal cancer and rectal polyps. This article discusses a transanal robotic surgery technique to perform full-thickness resections of benign and malignant rectal neoplasms. OBJECTIVE: The purpose of this study was to describe an initial experience with robotic transanal minimally invasive surgery. DESIGN: This was a retrospective cohort study of consecutive patients who underwent robotic transanal minimally invasive surgery. SETTINGS: The study was conducted at a high-volume colorectal surgery practice with a large health maintenance organization. PATIENTS: Patients at Southern California Kaiser Permanente with early rectal cancer and rectal polyps amenable to transanal excision were included. INTERVENTIONS: Transanal resection of rectal tumors were removed using robotic transanal minimally invasive surgery. MAIN OUTCOME MEASURES: Local recurrence of rectal pathology was measured. RESULTS: A total of 58 patients underwent robotic transanal minimally invasive surgery with full-thickness rectal resection by 4 surgeons for the following indications: rectal cancer (n = 28), rectal polyp (n = 18), rectal carcinoid (n = 11), and rectal GI stromal tumor (n = 1). Mean operative time was 66.2 minutes (range, 17-180 min). The mean tumor height from the anal verge was 8.8 cm (range, 4-14 cm), and the mean specimen size was 3.3 cm (range, 1.3-8.2 cm). A total of 57 (98.3%) of 58 specimens were intact, and 55 (94.8%) of 58 specimens had negative surgical margins. At a mean follow-up of 11.5 months (range, 0.3-33.3 mo), 3 patients (5.5%) developed local recurrences, and all underwent successful salvage surgery. LIMITATIONS: The study was limited by being a retrospective, nonrandomized trial with short follow-up. CONCLUSIONS: Robotic transanal minimally invasive surgery is a safe, oncologically effective surgical approach for rectal polyps and early rectal cancers. It offers the oncologic benefits and perioperative complication profile of other transanal minimally invasive surgical approaches but also enhances surgeon ergonomics and provides an efficient transanal rectal platform. See Video Abstract at http://links.lww.com/DCR/A759.

Efficacy of In Vitro Lithium Chloride Treatments on Dermacentor reticulatus.

Dermacentor reticulatus (Fabr., 1794) (Acari: Ixodidae) is parasite that spreads many diseases which are dangerous to humans and animals. Microelement lithium was found to have promising potential against the detrimental bee pest Varroa destructor. Furthermore, its effectiveness was confirmed against Dermanyssus gallinae, a major parasite of poultry, in vitro. In the present study, we investigated whether the efficacy of lithium chloride extends to other parasitic species, such as D. reticulatus. Our results revealed, for the first time, that the effectiveness of lithium chloride extends to D. reticulatus, confirmed to have 100% mortality at a relatively high minimum concentration of 1.38 M in vitro. The 24 h and 48 h median lethal concentration (LC50) values proved to be 0.654 M and 0.481 M, respectively, for this species. Our pilot study may contribute to a better understanding of the properties of lithium ion. Furthermore, it may elicit further studies aiming to reveal whether the different environmental mineral conditions may influence the D. reticulatus population. Further studies might reveal whether lithium has any possible veterinary relevance.

Genetic diversity and effect of temperature and pH on the growth of Macrophomina phaseolina isolates from sunflower fields in Hungary.

The effects of temperature and pH on the growth of 45 Hungarian Macrophomina phaseolina isolates from different locations and hosts were compared on the basis of their genetic diversity. One Spanish and two Serbian isolates were also included in the experiment. The most favourable temperature regimes for the development of the isolates ranged between 25 and 35 °C. The optimal pH for the pathogen varied between 4.0 and 6.0, but growth was observed on potato dextrose agar even at pH values of 3.0, 7.0 and 8.0. RAPD analysis with 13 different primer pairs generated 148 unambiguous bands. RFLP analysis involving 8 different restriction endonucleases was performed on a 1550 bp fragment of the rDNA region containing internal transcribed spacers (ITS1, ITS2), the 5.8S rDNA and part of the 25S rDNA. The greatest genetic distance values were obtained for three isolates, two from Hungary and one from Spain, which had similar values, but were quite distinct from all the others. A strong positive correlation was observed between the genetic distances and the growth parameters measured at various temperatures, and between the geographical data and the growth data sets at different pH values, but the correlation was less strong in the latter case. While Hungarian M. phaseolina populations are thought to reproduce clonally, the present results indicate the coexistence of different haplotypes in this area, and besides the geographical dominance of a given haplotype it was found that a closer genetic relationship might exist between spatially distinct haplotypes.

Leaf-spot disease on European mistletoe (Viscum album) caused by Phaeobotryosphaeria visci: a potential candidate for biological control.

Viscum album (European mistletoe), a perennial, evergreen, hemiparasitic shrub, infects a wide range of woody species. It adversely affects the height and diameter of growth and it is associated with increased mortality of its hosts. There is no effective control methods against it. We have found a specific hyperparasitic fungus, which can completely destroy European mistletoe by infecting its branches, leaves and berries. Both morphological and molecular identification, based on ribosomal internal transcribed spacer sequences (rDNA-ITS), established its identity as Phaeobotryosphaeria visci. Our analysis also revealed unexpected ITS variability, as compared to the previous studies, that needs to be considered in identifying of this pathogen. Because of its efficient pathogenicity this fungus might be a good candidate for biological control of mistletoe.

Comparison of Alternative Application Methods for Anti-Varroa Lithium Chloride Treatments.

Varroosis is one of the most dangerous threats to the bee industry but means of its treatment are still unsatisfactory. Lithium-based anti-Varroa treatments may provide an alternative, as this trace element can be a natural component of honey and is well tolerated by adult bees. However, it can be toxic to larvae and its use in beekeeping practice is not yet well understood. The present study aimed to investigate the efficacy of relevant application methods of acaricides used in beekeeping practice in brood-free conditions for lithium. Vaporisation proved to be an inefficient method of lithium treatment and killed only 9.9 ± 3.3% (mean ± SD) of mites in the hive. Lithium-impregnated paper strips showed moderate efficiency by killing 55.1 ± 26.2% of mites. The most effective way of applying lithium was the trickling method; different trickling treatments decreased the abundance of mites on average by 65 to 99.7%, depending on the applied dosage and the number of treatments. Repeated trickling treatments were more effective than single treatments, and they generally provided >90% efficiency. Experiments also proved that adding sugar to the trickling solution does not influence treatment efficiency. Thus, it is suggested that repeated and sugar-free trickling treatments with moderate lithium dosage could be the most rational methodology. Since lithium is not yet legalised in beekeeping practice, comprehensive studies are also needed to uncover the amount of lithium residue in bee products, depending on the treatment parameters.

Lithium Chloride Shows Effectiveness against the Poultry Red Mite (Dermanyssus gallinae).

The poultry red mite (Dermanyssus gallinae) is the main pest of poultry, causing severe problems by being a vector of several animal and human pathogens. The number of miticides is few, and their efficacy in practice implies problems of residues and resistance; therefore, the demand for a new and safe agent is constant. The present publication investigated the effectiveness of lithium chloride under in vitro conditions on poultry red mites. This chemical currently appears to be one of the most promising alternatives to study amongst potential applicants to treat varroosis, a fatal disease of honey bees. In Experiment I, the previously used experimental doses (5.52 M, 2.76 M, 1.38 M) on Varroa mites confirmed their in vitro activity on the poultry red mite. Three event times (uncontrolled movement, immobilisation and death) were recorded to base the response to treatment for each concentration. In Experiment II, the LD 50 value was calculated, i.e., the value at which 50% of the mites were killed by the treatment. This Experiment showed that the LD50 of lithium chloride = 0.265 M in the poultry red mite. It is to note that the study remained restricted to in vitro confirmation of lithium chloride's effectiveness on the parasite. Thus, further extensive studies are needed to decide whether it has any relevance in practice against D. gallinae, and also to assess potential residue problems that could affect poultry products.

Development of a simple PCR-based assay for the identification of triazine resistance in the noxious plant common ragweed (Ambrosia artemisiifolia) and its applicability in higher plants.

Bidirectional allele-specific PCR (Bi-PASA) was used to detect a point mutation causing triazine resistance in common ragweed (Ambrosia artemisiifolia). The external primers amplified a 278 bp standard DNA fragment in all genotypes. In the susceptible S264S genotypes, a 208 bp fragment was expected while in resistant S264G common ragweed genotypes a 109 bp band was expected. In resistant plants, both the wild and mutant type fragments were detected, indicating that the original triazine sensitive cpDNA is maintained in a heteroplasmic state in the resistant S264G genotypes. Additionally, in silico analysis confirmed the potential applicability of our diagnostic assay for other plant species. In 24 out of 74 taxa (32%), the assay could be used without any change, while in the others some of the primers should be redesigned before use.

Changes in Lithium Levels in Bees and Their Products Following Anti-Varroa Treatment.

The biggest threat to beekeeping is varroosis caused by the mite Varroa destructor. Chemicals available to treat this fatal disease may present problems of resistance or inconsistent efficacy. Recently, lithium chloride has appeared as a potential alternative. To date, the amount of residue lithium treatments may leave in honeybee products is poorly understood. Honeybees were fed with 25 mM lithiated sugar syrup, which was used in earlier studies. The accumulation and elimination of the lithium were monitored in bees and their products for 22 days. Lithium concentration increased in the entire body of the bees to day 4 post-treatment and then recovered rapidly to the control level. Lithium exposure was found to affect uncapped honey in the short term (<16 days), but ripe (capped) honey measured at the end of the trial remained affected. On the other hand, lithium treatment left beeswax lithium-free. Based on these data, we propose that comprehensive research on harvested honey is needed to decide on the veterinary use of lithium.

Contact Effect Contribution to the High Efficiency of Lithium Chloride Against the Mite Parasite of the Honey Bee.

Lithium chemicals have been proven to be very effective in eradicating Varroa destructor, the detrimental parasite of the honey bee; however, little is known about the side effects on brood and long term consequences on the colony. Earlier, it was proposed that the action mechanisms of lithium chloride do not include the contact mode. Here, we investigate this question using a paper strip test to demonstrate the concentration-dependent effectiveness of lithium in the contact mode of action, confirming that it is also a contact agent against the Varroa mite. According to our knowledge, this is the first report on the high varroicidal effect of lithium in the contact mode of action. Our findings may open up possibilities for novel ways of treatment (e.g., the use of lithiated strips) in the event that lithium salts become legal for use in apiculture.

Genome-Wide Identification of RNA Silencing-Related Genes and Their Expressional Analysis in Response to Heat Stress in Barley (Hordeum vulgare L.).

Barley (Hordeum vulgare L.) is an economically important crop cultivated in temperate climates all over the world. Adverse environmental factors negatively affect its survival and productivity. RNA silencing is a conserved pathway involved in the regulation of growth, development and stress responses. The key components of RNA silencing are the Dicer-like proteins (DCLs), Argonautes (AGOs) and RNA-dependent RNA polymerases (RDRs). Despite its economic importance, there is no available comprehensive report on barley RNA silencing machinery and its regulation. In this study, we in silico identified five DCL (HvDCL), eleven AGO (HvAGO) and seven RDR (HvRDR) genes in the barley genome. Genomic localization, phylogenetic analysis, domain organization and functional/catalytic motif identification were also performed. To understand the regulation of RNA silencing, we experimentally analysed the transcriptional changes in response to moderate, persistent or gradient heat stress treatments: transcriptional accumulation of siRNA- but not miRNA-based silencing factor was consistently detected. These results suggest that RNA silencing is dynamically regulated and may be involved in the coordination of development and environmental adaptation in barley. In summary, our work provides information about barley RNA silencing components and will be a ground for the selection of candidate factors and in-depth functional/mechanistic analyses.

An Alternative, High Throughput Method to Identify Csd Alleles of the Honey Bee.

Applying instrumental insemination in closely related honey bee colonies often leads to frequent lethality of offspring causing colony collapse. This is due to the peculiarities of honey bee reproductive biology, where the complementary sex determination (csd) gene drives sex determination within a haplodiploid system. Diploid drones containing homozygous genotypes are lethal. Tracking of csd alleles using molecular markers prevents this unwanted event in closed breeding programs. Our approach described here is based on high throughput sequencing (HTS) that provides more data than traditional molecular techniques and is capable of analysing sources containing multiple alleles, including diploid individuals as the bee queen. The approach combines HTS technique and clipping wings as a minimally invasive method to detect the complementary sex determiner (csd) alleles directly from honey bee queens. Furthermore, it might also be suitable for screening alleles of honey harvested from hives of a closed breeding facility. Data on alleles of the csd gene from different honey bee subspecies are provided. It might contribute to future databases that could potentially be used to track the origin of honey. With the help of tracking csd alleles, more focused crossings will be possible, which could in turn accelerate honey bee breeding programmes targeting increase tolerance against varroosis as well.

Mapping and DNA sequence characterisation of the Rysto locus conferring extreme virus resistance to potato cultivar 'White Lady'.

Virus resistance genes carried by wild plant species are valuable resources for plant breeding. The Rysto gene, conferring a broad spectrum of durable resistance, originated from Solanum stoloniferum and was introgressed into several commercial potato cultivars, including 'White Lady', by classical breeding. Rysto was mapped to chromosome XII in potato, and markers used for marker-assisted selection in breeding programmes were identified. Nevertheless, there was no information on the identity of the Rysto gene. To begin to reveal the identification of Rysto, fine-scale genetic mapping was performed which, in combination with chromosome walking, narrowed down the locus of the gene to approximately 1 Mb. DNA sequence analysis of the locus identified six full-length NBS-LRR-type (short NLR-type) putative resistance genes. Two of them, designated TMV2 and TMV3, were similar to a TMV resistance gene isolated from tobacco and to Y-1, which co-segregates with Ryadg, the extreme virus resistance gene originated from Solanum andigena and localised to chromosome XI. Furthermore, TMV2 of 'White Lady' was found to be 95% identical at the genomic sequence level with the recently isolated Rysto gene of the potato cultivar 'Alicja'. In addition to the markers identified earlier, this work generated five tightly linked new markers which can serve potato breeding efforts for extreme virus resistance.

Nitrogen utilization of potato genotypes and expression analysis of genes controlling nitrogen assimilation.

INTRODUCTION: Significant differences in nitrogen use efficiency (NUE) were detected previously among potato cultivars. Exploration of the genetic background may facilitate the breeding of cultivars with highly effective nitrogen use. METHODS: Expression of NUE genes was analyzed at three different N-supply levels in five potato genotypes. Correlations of NUE gene expressions and agronomical parameters with such indices as the nitrogen uptake efficiency, nitrogen utilization efficiency, NUE, and harvest indices were analyzed. RESULTS: The correlations between expression level of the nitrate-reductase, nitrite-reductase, ammonium transporter, and asparagine synthase genes and different agronomically important parameters were detected. DISCUSSION: Our results contribute to more rational, genotype-dependent nitrogen use in potato production and have relevance in breeding of new cultivars with better nitrogen utilization, as well as in production of seed potato.

Different expression pattern of flowering pathway genes contribute to male or female organ development during floral transition in the monoecious weed Ambrosia artemisiifolia L. (Asteraceae).

The highly allergenic and invasive weed Ambrosia artemisiifolia L. is a monoecius plant with separated male and female flowers. The genetic regulation of floral morphogenesis is a less understood field in the reproduction biology of this species. Therefore the objective of this work was to investigate the genetic control of sex determination during floral organogenesis. To this end, we performed a genome-wide transcriptional profiling of vegetative and generative tissues during the plant development comparing wild-growing and in vitro cultivated plants. RNA-seq on Illumina NextSeq 500 platform with an integrative bioinformatics analysis indicated differences in 80 floral gene expressions depending on photoperiodic and endogenous initial signals. Sex specificity of genes was validated based on RT-qPCR experiments. We found 11 and 16 uniquely expressed genes in female and male transcriptomes that were responsible particularly to maintain fertility and against abiotic stress. High gene expression of homologous such as FD, FT, TFL1 and CAL, SOC1, AP1 were characteristic to male and female floral meristems during organogenesis. Homologues transcripts of LFY and FLC were not found in the investigated generative and vegetative tissues. The repression of AP1 by TFL1 homolog was demonstrated in male flowers resulting exclusive expression of AP2 and PI that controlled stamen and carpel formation in the generative phase. Alterations of male and female floral meristem differentiation were demonstrated under photoperiodic and hormonal condition changes by applying in vitro treatments.

Temporary vascular shunts as initial treatment of proximal extremity vascular injuries during combat operations: the new standard of care at Echelon II facilities?

BACKGROUND: Historically, penetrating injuries to the extremities account for up to 75% of wounds sustained during combat and 10% of deaths. Rapid vascular control and perfusion of injured extremities at forward deployed Echelon II surgical facilities is essential to limit loss of life and maximize limb preservation. We review our experience with the management of extremity vascular trauma and report the largest single Echelon II experience to date on temporary vascular shunting (TVS) for proximal extremity vascular injuries. METHODS: Data on combat trauma patients presenting to a US Navy Echelon II forward surgical facility in Iraq were prospectively recorded during a 7-month period. Patients with suspected vascular injuries underwent exploration in the operating room. After vessel control, thrombectomy and instillation of heparinized saline, vascular injuries in the proximal extremity were temporarily shunted in a standardized fashion. Vascular injuries in the distal extremity were routinely ligated. After shunting, patients were transported to an Echelon III facility in the Iraqi Theater and underwent vascular reconstruction. They were followed through transfer to the Continental United States or discharge into the civilian Iraqi medical system. Shunt patency, limb salvage, and survival data were obtained by retrospective review of electronic medical records. RESULTS: Six hundred ten combat trauma patients were treated from August 16, 2006 to February 25, 2007. Thirty-seven patients (6.1%) sustained 73 injuries to major extremity vascular structures. Twenty-three proximal vascular shunts were placed in 16 patients with mean Injury Severity Score of 25 (range, 17-43) and mean mangled extremity severity score (MESS) of 8 (range, 5-10). Twenty-two of 23 shunts (95.6%) were patent upon arrival to the Echelon III facility and underwent successful autologous vein reconstruction. All shunt patients survived their injuries with 100% early limb preservation as followed through their first 30 days of medical care or discharge into the local medical community. CONCLUSIONS: Complex combat injuries to proximal extremity vessels should be routinely shunted at forward-deployed Echelon II facilities as part of the resuscitative, damage control process.

The No-go decay system degrades plant mRNAs that contain a long A-stretch in the coding region.

RNA quality control systems identify and degrade aberrant mRNAs, thereby preventing the accumulation of faulty proteins. Non-stop decay (NSD) and No-go decay (NGD) are closely related RNA quality control systems that act during translation. NSD degrades mRNAs lacking a stop codon, while NGD recognizes and decays mRNAs that contain translation elongation inhibitory structures. NGD has been intensively studied in yeast and animals but it has not been described in plants yet. In yeast, NGD is induced if the elongating ribosome is stalled by a strong inhibitory structure. Then, the mRNA is cleaved by an unknown nuclease and the cleavage fragments are degraded. Here we show that NGD also operates in plant. We tested several potential NGD cis-elements and found that in plants, unlike in yeast, only long A-stretches induce NGD. These long A-stretches trigger endonucleolytic cleavage, and then the 5' fragments are degraded in a Pelota-, HBS1- and SKI2- dependent manner, while XRN4 eliminates the 3' fragment. We also show that plant NGD operates gradually, the longer the A-stretch, the more efficient the cleavage. Our data suggest that mechanistically NGD is conserved in eukaryotes, although the NGD inducing cis-elements could be different. Moreover, we found that Arabidopsis AtPelota1 functions in both NGD and NSD, while AtPelota2 represses these quality control systems. The function of plant NGD will be discussed.

Expansion of Capsicum annuum fruit is linked to dynamic tissue-specific differential expression of miRNA and siRNA profiles.

Small regulatory RNAs, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) have emerged as important transcriptional and post-transcriptional regulators controlling a wide variety of physiological processes including fruit development. Data are, however, limited for their potential roles in developmental processes determining economically important traits of crops. The current study aimed to discover and characterize differentially expressed miRNAs and siRNAs in sweet pepper (Capsicum annuum) during fruit expansion. High-throughput sequencing was employed to determine the small regulatory RNA expression profiles in various fruit tissues, such as placenta, seed, and flesh at 28 and 40 days after anthesis. Comparative differential expression analyses of conserved, already described and our newly predicted pepper-specific miRNAs revealed that fruit expansion is accompanied by an increasing level of miRNA-mediated regulation of gene expression. Accordingly, ARGONAUTE1 protein, the primary executor of miRNA-mediated regulation, continuously accumulated to an extremely high level in the flesh. We also identified numerous pepper-specific, heterochromatin-associated 24-nt siRNAs (hetsiRNAs) which were extremely abundant in the seeds, as well as 21-nt and 24-nt phased siRNAs (phasiRNAs) that were expressed mainly in the placenta and the seeds. This work provides comprehensive tissue-specific miRNA and siRNA expression landscape for a developing pepper fruit. We identified several novel, abundantly expressing tissue- and pepper-specific small regulatory RNA species. Our data show that fruit expansion is associated with extensive changes in sRNA abundance, raising the possibility that manipulation of sRNA pathways may be employed to improve the quality and quantity of the pepper fruit.

Correction: Expansion of Capsicum annum fruit is linked to dynamic tissue-specific differential expression of miRNA and siRNA profiles.

[This corrects the article DOI: 10.1371/journal.pone.0200207.].