A transmission assay of 'Candidatus Liberibacter asiaticus' using citrus phloem sap and topical feeding to its insect vector, Diaphorina citri.

'Candidatus Liberibacter asiaticus', the putative causal agent of citrus greening disease is transmitted by the Asian citrus psyllid, Diaphorina citri in a propagative, circulative, and persistent manner. Unfortunately, 'Ca. L. asiaticus' is not yet available in pure culture to carry out Koch's postulates and to confirm its etiology. When a pure culture is available, an assay to test its infectivity in both the insect vector and the plant host will be crucial. Herein, we described a transmission assay based on the use of phloem sap extracted from infected citrus plants and topical feeding to D. citri nymphs. Phloem sap was collected by centrifugation, diluted with 0.1 M phosphate buffer pH 7.4 containing 20% (w/v) sucrose and 0.1% ascorbic acid (w/v) as an antioxidant, and delivered to third through fifth instar nymphs by placing droplets on the mouthparts. Nymphs unfolded the stylets and acquired the phloem sap containing the bacterial pathogen. Nymphs were then placed onto Citrus macrophylla seedlings (ten nymphs/seedling) for an inoculation period of two weeks. A transmission rate of up to 80% was recorded at six months post-inoculation. The method could be a powerful tool to test the transmissibility of the bacterial pathogen after various treatments to reduce the viability of the bacteria or to block its transmission. In addition, it might be a potent assay to achieve Koch's postulates if a pure culture of 'Ca. L. asiaticus' becomes available.

Genomic signatures of geographic isolation and natural selection in coral reef fishes.

The drivers of speciation remain among the most controversial topics in evolutionary biology. Initially, Darwin emphasized natural selection as a primary mechanism of speciation, but the architects of the modern synthesis largely abandoned that view in favour of divergence by geographic isolation. The balance between selection and isolation is still at the forefront of the evolutionary debate, especially for the world's tropical oceans where biodiversity is high, but isolating barriers are few. Here, we identify the drivers of speciation in Pacific reef fishes of the genus Acanthurus by comparative genome scans of two peripheral populations that split from a large Central-West Pacific lineage at roughly the same time. Mitochondrial sequences indicate that populations in the Hawaiian Archipelago and the Marquesas Islands became isolated approximately 0.5 Ma. The Hawaiian lineage is morphologically indistinguishable from the widespread Pacific form, but the Marquesan form is recognized as a distinct species that occupies an unusual tropical ecosystem characterized by upwelling, turbidity, temperature fluctuations, algal blooms and little coral cover. An analysis of 3737 SNPs reveals a strong signal of selection at the Marquesas, with 59 loci under disruptive selection including an opsin Rh2 locus. While both the Hawaiian and Marquesan populations indicate signals of drift, the former shows a weak signal of selection that is comparable with populations in the Central-West Pacific. This contrast between closely related lineages reveals one population diverging due primarily to geographic isolation and genetic drift, and the other achieving taxonomic species status under the influence of selection.

Silencing of δ-aminolevulinic acid dehydratase via virus induced gene silencing promotes callose deposition in plant phloem.

The δ-aminolevulinic acid dehydratase (ALAD) enzyme is an intermediate in the biosynthetic pathway of tetrapyrroles. It combines two δ-aminolevulinic acid (δ-ALA) molecules to form the pyrrole, porphobilinogen, an important precursor for plant pigments involved in photosynthesis, respiration, light-sensing, and nutrient uptake. Our recent efforts showed that, in citrus, silencing of ALAD gene via Citrus tristeza virus-induced gene silencing, caused yellow spots and necrosis in leaves and in developing new shoots. Silencing of ALAD gene reduced leaf pigments and altered leaf metabolites. Moreover, total phenolic content, H2O2, and reactive oxygen species (ROS) increased, indicating that silencing of ALAD induced severe stress. Herein, we hypothesized that conditions including lower sucrose, elevated ROS, alteration of microRNA involved in RNAi regulatory protein Argonaute 1 (AGO1) and ROS lead to higher deposition of callose in phloem tissues. Using aniline blue staining and gene expression analysis of callose synthases, we showed significant deposition of callose in ALAD-silenced citrus.

Biosafety Practices When Working with Bats: A Guide to Field Research Considerations.

Introduction: Field work with bats is an important contribution to many areas of research in environmental biology and ecology, as well as microbiology. Work with bats poses hazards such as bites and scratches, and the potential for exposure to infectious pathogens such as rabies virus. It also exposes researchers to many other potential hazards inherent to field work, such as environmental conditions, delayed emergency responses, or challenging work conditions. Methods: This article discusses the considerations for a thorough risk assessment process around field work with bats, pre- and post-occupational health considerations, and delves into specific considerations for areas related to biosafety concerns-training, personal protective equipment, safety consideration in field methods, decontamination, and waste. It also touches on related legal and ethical issues that sit outside the realm of biosafety, but which must be addressed during the planning process. Discussion: Although the focal point of this article is bat field work located in northern and central America, the principles and practices discussed here are applicable to bat work elsewhere, as well as to field work with other animal species, and should promote careful considerations of how to safely conduct field work to protect both researchers and animals.

Influence of Rootstock on the Leaf Volatile Organic Compounds of Citrus Scion Is More Pronounced after the Infestation with Diaphorina citri.

Nowadays, citrus greening or Huanglongbing is considered the most destructive disease in the citrus industry worldwide. In the Americas and Asia, the disease is caused by the putative pathogen, 'Candidatus Liberibacter asiaticus' and transmitted by the psyllid vector, Diaphorina citri. It has been shown that volatile organic compounds (VOC) that are released from citrus leaves attract the psyllid vector. Herein, we tested whether the rootstock influenced the stored VOC profile in the scion leaves and if these influences were altered after infestation with D. citri. The VOC profiles of the hexane-extracted leaves of the mandarin hybrid 'Sugar Belle' that were grafted on three different rootstocks (C-35, sour orange (SO), and US-897) with and without infestation with D. citri were studied. The GC-MS analysis showed that the scion VOC profiles of the non-infested control trees were similar to each other, and rootstock was not a strong influence. However, after one month of infestation with D. citri, clear differences in the scion VOC profiles appeared that were rootstock dependent. Although the total scion leaf VOC content did not differ between the three rootstocks, the infestation increased scion monoterpenes significantly on US-897 and C-35 rootstock, increased terpene alcohols on US-897 and SO rootstock, and increased sesquiterpenes on SO. Infestation with D. citri significantly reduced fatty acids and fatty acid esters across all of the rootstocks. Therefore, our results suggest that rootstock choice could influence scions with an inducible volatile defense by enhancing the amounts of VOCs that are available for repelling vectors or for signaling to their natural enemies or parasitoids. According to this study, US-897 may be the best choice among the three that were studied herein, due to its diverse and robust VOC defense response to infestation with D. citri.

Size of the direct-to-consumer genomic testing market.

There has been enormous interest in the recent development of consumer genomics services, but very little is known about their impact. Using publicly available information, we estimate that the market for genetic susceptibility tests for complex diseases is much smaller than previously suggested, and hence consider that regulation through restrictive statutory legislation may be excessive.

Opicapone and Levodopa-Carbidopa Intestinal Gel Infusion: The Way Forward Towards Cost Savings for Healthcare Systems?

Combined catechol-O-methyl-transferase-inhibition and Levodopa-Carbidopa intestinal gel (LCIG) infusion has the potential to reduce LCIG daily dose and the costs of this therapy. In this retrospective analysis, we report on Parkinson's disease (PD) patients on LCIG with concomitant Opicapone. In 11 patients, the introduction of Opicapone led to LCIG daily dose being reduced by 24.8% (p = 0.05) without any significant worsening of dyskinesia. Three patients withdrew from Opicapone due to side effects or inefficacy. LCIG daily dose reduction could lead to cost savings of £142,820.63/year in the United Kingdom while maintaining clinical care.

Effect of Adjuvants on Oxytetracycline Uptake upon Foliar Application in Citrus.

Recently in Florida, foliar treatments using products with the antibiotics oxytetracyclineand streptomycin have been approved for the treatment of citrus Huanglongbing (HLB), which iscaused by the putative bacterial pathogen 'Candidatus Liberibacter asiaticus'. Herein, we assessedthe levels of oxytetracycline and 'Ca. L. asiaticus' titers in citrus trees upon foliar applications withand without a variety of commercial penetrant adjuvants and upon trunk injection. The level ofoxytetracycline in citrus leaves was measured using an oxytetracycline ELISA kit and 'Ca. L.asiaticus' titer was measured using quantitative PCR. Low levels of oxytetracycline were taken upby citrus leaves after foliar sprays of oxytetracycline in water. Addition of various adjuvants to theoxytetracycline solution showed minimal effects on its uptake by citrus leaves. The level ofoxytetracycline in leaves from trunk-injected trees was higher than those treated with all foliarapplications. The titer of 'Ca. L. asiaticus' in the midrib of leaves from trees receiving oxytetracyclineby foliar application was not affected after four days and thirty days of application, whereas thetiter was significantly reduced in oxytetracycline-injected trees thirty days after treatment.Investigation of citrus leaves using microscopy showed that they are covered by a thick lipidizedcuticle. Perforation of citrus leaf cuticle with a laser significantly increased the uptake ofoxytetracycline, decreasing the titer of 'Ca. L. asiaticus' in citrus leaves upon foliar application.Taken together, our findings indicate that trunk injection is more efficient than foliar spray evenafter the use of adjuvants. Our conclusion could help in setting useful recommendations for theapplication of oxytetracycline in citrus to improve tree health, minimize the amount of appliedantibiotic, reduce environmental exposure, and limit off-target effects.

Metabolic Profiling of Hybrids Generated from Pummelo and Citrus latipes in Relation to Their Attraction to Diaphorina citri, the Vector of Huanglongbing.

The citrus industry at present is severely affected by huanglongbing disease (HLB). HLB is caused by the supposed bacterial pathogen "Candidatus Liberibacter asiaticus" and is transmitted by the insect vector, the Asian citrus psyllid, Diaphorina citri Kuwayama. Developing new citrus hybrids to improve HLB management is much needed. In this study, we investigated the metabolomic profiles of three new hybrids produced from the cross of C2-5-12 Pummelo (Citrus maxima (L.) Osbeck) × pollen from Citrus latipes. The hybrids were selected based on leaf morphology and seedling vigor. The selected hybrids exhibited compact and upright tree architecture as seen in C. latipes. Hybrids were verified by simple sequence repeat markers, and were subjected to metabolomic analysis using gas chromatography-mass spectrometry. The volatile organic compounds (VOCs) and polar metabolites profiling also showed that the new hybrids were different from their parents. Interestingly, the levels of stored VOCs in hybrid II were higher than those observed in its parents and other hybrids. The level of most VOCs released by hybrid II was also higher than that released from its parents. Additionally, the preference assay showed that hybrid II was more attractive to D. citri than its parents and other hybrids. The leaf morphology, compact and upright architecture of hybrid II, and its attraction to D. citri suggest that it could be used as a windbreak and trap tree for D. citri (double duty), once its tolerance to HLB disease is confirmed. Our results showed that metabolomic analysis could be successfully used to understand the biochemical mechanisms controlling the interaction of D. citri with its host plants.

Metabolic alterations in the nymphal instars of Diaphorina citri induced by Candidatus Liberibacter asiaticus, the putative pathogen of huanglongbing.

Currently, huanglongbing is the most damaging disease of citrus causing huge economic losses. The disease is caused by the Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). The pathogen is transmitted in a persistent propagative circulative manner within its vector, the Asian citrus psyllid, Diaphorina citri. Exploring the metabolic alteration in the vector may lead to a better understanding of the nutritional needs of CLas and to designing an artificial medium for culturing the pathogen. It has been shown that the nymphal stages have a greater role in transmission mainly because they feed on plants more actively than adults. In this study, we carried out an untargeted comparative metabolomic analysis for healthy and CLas-infected 4th / 5th instar nymphs. The metabolic analysis was performed using trimethylsilylation and methyl chloroformate derivatization followed by Gas Chromatography-Mass Spectrometry (GC-MS). Overall, the changes in the nymph metabolism due to the infection with CLas were more pronounced than in adults, as we previously published. Nymphs reared on CLas-infected Valencia sweet orange were higher in many metabolites, mainly those of the TCA cycle, C16 and C18 fatty acids, glucose, sucrose, L-proline, L-serine, pyroglutamic acid, saccharic acid, threonic acid and myo-inositol than those reared on healthy plants. In contrast, CLas-infected nymphs were lower in putrescine, glycine, L -phenylalanine, L -tyrosine, L -valine, and chiro-inositol. The information provided from this study may contribute in acceleration of the availability of CLas in culture and consequent screening of antibacterial compounds to discover a definitive solution for huanglongbing.

Effect of different rootstocks on the leaf metabolite profile of 'Sugar Belle' mandarin hybrid.

Currently, citrus greening is threatening the citrus industry worldwide. So far, there is no effective cure for this destructive disease and management mainly depends on the control of Diaphorina citri vector using insecticides. Although the use of different rootstocks could increase citrus scions' tolerance to biotic and abiotic stresses, little work has been conducted to investigate the effect of rootstocks on citrus tolerance to citrus greening pathogen. In this study, we investigated the effect of rootstock on the metabolite profile of 'Sugar Belle' mandarin hybrid using gas-chromatography mass spectrometry (GC-MS). The principle component analysis showed that the metabolite profiles of the 'Sugar Belle' mandarin hybrid on the three selected rootstocks were different from each other. These results indicated that rootstocks could affect the primary and secondary metabolites of citrus scions, and consequently could affect scion tolerance to pathogens.

All roads lead to Rome: Towards understanding different avenues of tolerance to huanglongbing in citrus cultivars.

Citrus tolerance to huanglongbing could result from tolerance to the pathogen Candidatus Liberibacter asiaticus (CLas) and/or to its vector Diaphorina citri. Field observations and greenhouse-controlled studies showed that some citrus cultivars were more tolerant than others. However, the mechanism(s) behind the tolerance has not been determined yet. Using GC-MS, we investigated the volatile organic compounds (VOCs) and the non-volatile metabolite profiles of two tolerant citrus cultivars- Australian finger lime, 'LB8-9' Sugar Belle® mandarin hybrid, and a recently released mandarin hybrid 'Bingo'. The three were grafted onto the rootstock, Carrizo citrange. Our findings showed that the metabolomic profiles of Australian finger lime were different from that of 'LB8-9'. Finger lime was high in many amino acids and tricarboxylic acid intermediates, whereas 'LB8-9' was high in several amino acids, sugars, and sugar alcohols. 'LB8-9' was high in thymol, which is known for its strong antimicrobial activity against a panel of pathogenic bacteria. The metabolomic profiles of 'Bingo' were intensely different from the other mandarin hybrid, 'LB8-9', including a reduced thymol biosynthetic pathway and low amounts of most of the amino acids and sugar alcohols. Remarkably, 1,8-cineole (eucalyptol) was only detected in 'Bingo', indicating that eucalyptol could have feeding and ovipositional repellency against D. citri. The metabolite profiles generated for HLB-tolerant citrus species will improve the ability of citrus breeders and will allow them to take more informed decisions. Metabolomic profiling of HLB-tolerant citrus species could identify tolerance specific markers that can be introduced to other commercial citrus cultivars to improve their tolerance to HLB disease.

Insulin Matters: A Practical Approach to Basal Insulin Management in Type 2 Diabetes.

It is currently estimated that 11 million Canadians are living with diabetes or prediabetes. Although hyperglycemia is associated with serious complications, it is well established that improved glycemic control reduces the risk of microvascular complications and can also reduce cardiovascular (CV) complications over the long term. The UKPDS and ADVANCE landmark trials have resulted in diabetes guidelines recommending an A1C target of ≤ 7.0% for most patients or a target of ≤ 6.5% to further reduce the risk of nephropathy and retinopathy in those with type 2 diabetes (T2D), if it can be achieved safely. However, half of the people with T2D in Canada are not achieving these glycemic targets, despite advances in diabetes pharmacological management. There are many contributing factors to account for this poor outcome; however, one of the major factors is the delay in treatment advancement, particularly a resistance to insulin initiation and intensification. To simplify the process of initiating and titrating insulin in T2D patients, a group of Canadian experts reviewed the evidence and best clinical practices with the goal of providing guidance and practical recommendations to the diabetes healthcare community at large. This expert panel included general practitioners (GPs), nurses, nurse practitioners, endocrinologists, dieticians, pharmacists, and a psychologist. This article summarizes the panel recommendations.

Profiling of volatile organic compounds released from individual intact juvenile and mature citrus leaves.

Plants release volatiles to communicate with each other and to attract or repel insects. The methods used to collect volatiles are varied. Here, we describe a simple solvent-less, solid phase microextraction-based method to collect the volatiles released from intact citrus leaves. We were able to collect up to 39 volatiles from both juvenile and mature leaves. Our results indicated that juvenile leaves produced both monoterpenes and sesquiterpenes, and while mature leaves continued to produce a variety of monoterpenes, their release of sesquiterpenes decreased dramatically. The finding that juvenile leaves emitted higher levels of sesquiterpenes while mature leaves released mostly monoterpenes suggests that younger leaves of plants may be involved in a more complex chemical communication system.

Metabolically speaking: Possible reasons behind the tolerance of 'Sugar Belle' mandarin hybrid to huanglongbing.

Huanglongbing (HLB) is currently considered the most destructive disease of citrus. Since its spread to the Americas, HLB has killed millions of trees and caused a sharp decline in production in many citrus growing regions. With the continuous spread of HLB disease in Florida and worldwide, there is an urgent need for the development of commercial citrus cultivars with a strong tolerance to HLB. Interestingly, field observations showed that some of the recently released mandarin hybrids such as 'Sugar Belle' were tolerant to HLB. In this study, we investigated the volatile and non-volatile metabolites of greenhouse-grown 'Sugar Belle' mandarin and four of its ancestors in order to understand why 'Sugar Belle' mandarin is relatively tolerant to HLB. Leaf volatiles were directly extracted with hexane and analyzed using gas chromatography-mass spectrometry (GC-MS). Leaf polar metabolites were extracted with a mixture of methanol:water (1:1, v/v), derivatized to their trimethylsilyl ethers, and analyzed using GC-MS. Forty-seven volatile compounds and forty-two polar metabolites were detected in 'Sugar Belle' mandarin leaves and its ancestors. 'Sugar Belle' was high in several volatiles such as α-thujene, para-cymene, γ-terpinene, thymol, ß-elemene, and (E)-ß-caryophyllene. Some of these volatiles, especially thymol, ß-elemene, and (E)-ß-caryophyllene are known for their anti-microbial activity. In addition, 'Sugar Belle' mandarin was the highest in synephrine, benzoic acid, ferulic acid, caffeic acid, chiro-inositol, fructose, glucose, threonic acid, saccharic acid, and galactaric acid, and the second in threonine, malic acid, and myo-inositol compared to the ancestors. Phenolic compounds such as benzoic, ferulic, and caffeic acids may act as antibacterial agents, whereas others like sugar alcohols may protect 'Sugar Belle' mandarin from stress during pathogen attack. The tolerance of 'Sugar Belle' and other newly released mandarin hybrids should be further evaluated using greenhouse controlled studies. If tolerance of these hybrids is confirmed, they could be used to replace the traditionally susceptible cultivars.

Reducing harm from falls.

Serious adverse event reporting from district health boards (DHBs) brought in-hospital falls to the attention of the Health Quality & Safety Commission (the Commission) when it was incepted in 2010. In 2012, responding to the large numbers reported, the Commission began planning for a three-year programme to reduce harm from falls, initially to run 2013-2015. In this article we discuss the serious consequences of falls, and the challenges and practical considerations involved in reducing the risk of falling and the rate of falls. We explore the Commission's choice of an adaptive approach in its programme, and show how a targeted measurement framework and national action has led to a nationwide statistically significant reduction in fractured neck of femur (hip fracture) and associated costs resulting from in-hospital falls, from a median of 12 per 100,000 admissions to eight per 100,000 admissions, sustained as at June 2016 for six quarters. This reduction reflects nationwide implementation of two key care processes: 1.) the percentage of patients 75 and over provided with an assessment of their risk of falling upon admission to hospital has risen from 77% in the first quarter of 2013 to 91% nationally in June 2016, 2.) the percentage of those with identified risk who were provided with an individualised care plan that addressed those risks has risen from 77% of older patients in the first quarter of 2013 to 95% nationally in June 2016. (These results are also reflected in a 14% decrease to 30 June 2016 in numbers of falls reported by DHBs as serious adverse events). Finally, we give a call to arms to the disparate health practitioners and services across all settings for individualised responses to prevent falls one patient at a time, and for leadership responses that promote an integrated approach to falls in older people.