Evidence of the biochemical basis of host virulence in the greenbug aphid, Schizaphis graminum (Homoptera: Aphididae).

Biotypes of aphids and many other insect pests are defined based on the phenotypic response of host plants to the insect pest without considering their intrinsic characteristics and genotypes. Plant breeders have spent considerable effort developing aphid-resistant, small-grain varieties to limit insecticide control of the greenbug, Schizaphis graminum. However, new S. graminum biotypes frequently emerge that break resistance. Mechanisms of virulence on the aphid side of the plant-insect interaction are not well understood. S. graminum biotype H is highly virulent on most small grain varieties. This characteristic makes biotype H ideal for comparative proteomics to investigate the basis of biotype virulence in aphids. In this study, we used comparative proteomics to identify protein expression differences associated with virulence. Aphid proteins involved in the tricarboxylic acid cycle, immune system, cell division, and antiapoptosis pathways were found to be up-regulated in biotype H relative to other biotypes. Proteins from the bacterial endosymbiont of aphids were also differentially expressed in biotype H. Guided by the proteome results, we tested whether biotype H had a fitness advantage compared with other S. graminum biotypes and found that biotype H had a higher reproductive fitness as compared with two other biotypes on a range of different wheat germplasms. Finally, we tested whether aphid genetics can be used to further dissect the genetic mechanisms of biotype virulence in aphids. The genetic data showed that sexual reproduction is a source of biotypic variation observed in S. graminum.

Biomarker discovery from the top down: Protein biomarkers for efficient virus transmission by insects (Homoptera: Aphididae) discovered by coupling genetics and 2-D DIGE.

Yellow dwarf viruses cause the most economically important virus diseases of cereal crops worldwide and are vectored by aphids. The identification of vector proteins mediating virus transmission is critical to develop sustainable virus management practices and to understand viral strategies for circulative movement in all insect vectors. Previously, we applied 2-D DIGE to an aphid filial generation 2 population to identify proteins correlated with the transmission phenotype that were stably inherited and expressed in the absence of the virus. In the present study, we examined the expression of the DIGE candidates in previously unstudied, field-collected aphid populations. We hypothesized that the expression of proteins involved in virus transmission could be clinically validated in unrelated, virus transmission-competent, field-collected aphid populations. All putative biomarkers were expressed in the field-collected biotypes, and the expression of nine of these aligned with the virus transmission-competent phenotype. The strong conservation of the expression of the biomarkers in multiple field-collected populations facilitates new and testable hypotheses concerning the genetics and biochemistry of virus transmission. Integration of these biomarkers into current aphid-scouting methodologies will enable rational strategies for vector control aimed at judicious use and development of precision pest control methods that reduce plant virus infection.

Biotypic diversity in greenbug (Hemiptera: Aphididae): microsatellite-based regional divergence and host-adapted differentiation.

Nineteen isolates of the cereal aphid pest greenbug, Schizaphis graminum (Rondani) (Hemiptera: Aphididae), were collected from wheat, Triticum aestivum L.; barley, Hordeum vulgare L.; or noncultivated grass hosts in five locations from Colorado and Wyoming. Parthenogenetic colonies were established. Biotypic profiles of the 19 isolates were determined based on their abilities to damage a set of host plant differentials, and 13 new biotypes were identified. Genetic diversity among the 19 isolates and five previously designated greenbug biotypes (E, G, H, I, and K) was examined with 31 cross-species transferable microsatellite (simple sequence repeat) markers. Neighbor-joining clustering analysis of marker data revealed host-adapted genetic divergence as well as regional differentiation of greenbug populations. Host associated biotypic variation seems to be more obvious in "agricultural biotypes," whereas isolates collected from noncultivated grasses tend to show more geographic divergence. It seems that the biotype sharing the most similar biotypic profiles and the same geographic region with current prevailing one may have the greatest potential to become the new prevailing biotype. Close monitoring of greenbug population dynamics especially biotypic variation on both crop plants and noncultivated grasses in small grain production areas may be a useful strategy for detecting potentially new prevailing virulent biotypes of the greenbug.

Host associations and incidence of Diuraphis spp. in the Rocky Mountain region of the United States, and pictorial key for their identification.

The Russian wheat aphid, Diuraphis noxia Kurdjumov, is an introduced species first identified in 1986 into the United States. It has since become a major pest of wheat, Triticum aestivum L., and other small grains in the western United States. Three other Diuraphis species, Diuraphis frequens (Walker), Diuraphis mexicana (McVicar Baker), and Diuraphis tritici (Gillette), were already endemic to the United States before the introduction of D. noxia. The objective of this study was to determine the occurrence and host associations of these four Diuraphis spp. in the Rocky Mountain region that borders the western Great Plains to better understand their distribution and ecological interactions. In addition, a key to these species with photographs of live or fresh preparations of specimens is presented to aid in their identification. D. noxia was the most widely distributed species in the study area spanning the Rocky Mountain areas of Wyoming, New Mexico, Utah, and Colorado. This species was most common in the cereal-producing areas of the Colorado Plateau ecoregion. D. frequens was found to be the predominant species in the Alpine/Aspen Mountain areas of the South Central Rockies and Colorado Rockies ecoregions. The other Diuraphis species were rarely encountered even though their plant hosts occurred in the ecoregions sampled. D. noxia shared common hosts and was found co-infesting grasses with other Diuraphis species. Therefore, the potential exists for D. noxia to impact the other native Diuraphis species.

Effects of Lysulin™ supplementation on pre-diabetes: study protocol for a randomized controlled trial.

BACKGROUND: Diabetes mellitus is rapidly becoming one of the leading causes of morbidity and mortality worldwide. Preventive measures have become important, especially in the South Asian region and other parts of the world where diabetes is becoming increasingly prevalent. We postulate that a product containing amino acid lysine, micronutrient zinc and vitamin C will have beneficial effects on glycemic control and disease progression in patients with pre-diabetes and we aim to evaluate this hypothesis using a phase II/III randomized double-blind controlled clinical trial design. METHODS/DESIGN: The study will be conducted as a randomized, double-blind, placebo-controlled clinical trial for a period of 6 months. The study has been approved by the Ethics Review Committee of Faculty of Medicine, University of Colombo, Sri Lanka. A total of 110 adults with pre-diabetes will be recruited for the study. They will be randomly assigned to the test and placebo groups on a 1:1 ratio. The test group will receive two tablets of Lysulin™ three times a day and the control group will receive identical placebo tablets. The study drugs will be double blinded to both investigators and subjects. The visits and the evaluations will be done as follows: screening (visit 0), 1 month (visit 1), 3 months (visit 2) and 6 months (visit 4). The primary outcome will be defined as change in glycemic control measured by HbA1c from baseline. Data will be analyzed using SPSS v16. DISCUSSION: To our knowledge this is one of the first randomized controlled trials evaluating the effects of Lysulin™ (a combination of lysine, vitamin C and zinc) in pre-diabetes. This study will provide the necessary groundwork for future large-scale multicentered clinical trials. The result, positive or negative, should provide a step change in the evidence guiding current and future policies regarding management of pre-diabetes. TRIAL REGISTRATION: Sri Lanka Clinical Trials Registry, SLCTR/2018/022 . Registered on 13 July 2018. Study protocol version 2.0 (23 March 2018).

Distribution and diversity of russian wheat aphid (Hemiptera: Aphididae) biotypes in North America.

Wheat, Triticum aestivum L., with Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae) resistance based on the Dn4 gene has been important in managing Russian wheat aphid since 1994. Recently, five biotypes (RWA1-RWA5) of this aphid have been described based on their ability to differentially damage RWA resistance genes in wheat. RWA2, RWA4, and RWA5 are of great concern because they can kill wheat with Dn4 resistance. In 2005, 365 Russian wheat aphid clone colonies were made from collections taken from 98 fields of wheat or barley, Hordeum vulgare L., in Oklahoma, Texas, New Mexico, Colorado, Kansas, Nebraska, and Wyoming to determine their biotypic status. The biotype of each clone was determined through its ability to differentially damage two resistant and two susceptible wheat entries in two phases of screening. The first phase determined the damage responses of Russian wheat aphid wheat entries with resistance genes Dn4, Dn7, and susceptible 'Custer' to infestations by each clone to identify RWA1 to RWA4. The second phase used the responses of Custer and 'Yuma' wheat to identify RWA1 and RWA5. Only two biotypes, RWA1 and RWA2, were identified in this study. The biotype composition across all collection sites was 27.2% RWA1 and 72.8% RWA2. RWA biotype frequency by state indicated that RWA2 was the predominant biotype and composed 73-95% of the biotype complex in Texas, Oklahoma, Colorado, and Wyoming. Our study indicated that RWA2 is widely distributed and that it has rapidly dominated the biotype complex in wheat and barley within its primary range from Texas to Wyoming. Wheat with the Dn4 resistance gene will have little value in managing RWA in the United States, based on the predominance of RWA2.

Physiological and biochemical responses of resistant and susceptible wheat to injury by Russian wheat aphid.

We examined the physiological and biochemical responses of resistant ('Halt' and 'Prairie Red') and susceptible ('TAM 107') wheat, Triticum aestivum L., to injury by the Russian wheat aphid, Diuraphis noxia (Mordvilko). Photosynthetic capacity was evaluated by measuring assimilation/internal CO2 (A/Ci) curves, chlorophyll fluorescence, chlorophyll, and nonstructural carbohydrate content. Total protein and peroxidase specific activity also were determined. No significant differences were detected in chlorophyll concentration between aphid-infested and control TAM 107 plants. The aphid-infested resistant cultivars had similar or significantly higher chlorophyll concentrations compared with their respective control plants. Measurements over time showed that infested Halt plants had delays in photosynthetic senescence, Prairie Red plants had photosynthetic rate changes that were similar to control plants, and TAM 107 plants displayed accelerated photosynthetic senescence patterns. The photochemical and nonphotochemical quenching coefficients were significantly higher in infested Halt plants compared with their respective control plants on day 3. Infested TAM 107 plants had significantly higher photochemical quenching compared with control plants at all times evaluated, and they had significantly higher nonphotochemical quenching on day 3. Throughout the experiment, infested Prairie Red plants exhibited photochemical and nonphotochemical quenching coefficient values that were not significantly different from control plants. Total protein content was not significantly different between aphid-infested and control plants for all cultivars. Differences between physiological responses of infested susceptible and resistant cultivars, particularly temporal changes in photosynthetic activity, imply that resistant Halt and Prairie Red wheat tolerate some impacts of aphid injury on photosynthetic integrity.

Biotypic diversity in greenbug (Hemiptera: Aphididae): characterizing new virulence and host associations.

Biotypic diversity of the greenbug, Schizaphis graminum (Rondani) (Hemiptera: Aphididae), was assessed among populations collected from cultivated wheat, Triticum aestivum L., and sorghum, Sorghum bicolor (L.) Moench, and their associated noncultivated grass hosts. Greenbugs were collected during May through August 2002 from 30 counties of Kansas, Nebraska, Oklahoma, and Texas. Discounting the presumptive biotype A, five of the remaining nine letter-designated greenbug biotypes were collected; however, biotypes C, F, J, and K were not detected. Biotypes E and I exhibited the greatest host range and were the only biotypes collected in all four states. Sixteen greenbug clones, collected from eight plant species, exhibited unique biotype profiles. Eleven were collected from noncultivated grasses, three from wheat, and two from sorghum. The most virulent biotypes were collected from noncultivated hosts. The great degree of biotypic diversity among noncultivated grasses supports the contention that the greenbug species complex is composed of host-adapted races that diverged on grass species independently of, and well before, the advent of modern agriculture.

Biotypic variation among north American Russian wheat aphid (Homoptera: Aphididae) populations.

The Russian wheat aphid, Diuraphis noxia (Mordvilko) (Homoptera: Aphididae), has been a major economic pest of small grains in the western United States since its introduction in 1986. Recently, a new Russian wheat aphid biotype was discovered in southeastern Colorado that damaged previously resistant wheat, Triticum aestivum L. Biotype development jeopardizes the durability of plant resistance, which has been a cornerstone for Russian wheat aphid management. Our objective was to assess the relative amount of biotypic diversity among Russian wheat aphid populations collected from cultivated wheat and barley, Hordeum vulgare L. We conducted field surveys from May through June 2002 and August 2003 from seven counties within Texas, Kansas, Nebraska, and Wyoming. Based upon a foliar chlorosis damage rating, three new Russian wheat aphid biotypes were identified, one of which was virulent to all characterized sources of Russian wheat aphid resistance. The future success of Russian wheat aphid resistance breeding programs will depend upon the continual monitoring of extant biotypic diversity and determination of the ecological and genetic factors underlying the development of Russian wheat aphid biotypes.

Virus Transmission Phenotype Is Correlated with Host Adaptation Among Genetically Diverse Populations of the Aphid Schizaphis graminum.

ABSTRACT Schizaphis graminum is an important insect pest of several grain crops and an efficient vector of cereal-infecting luteoviruses and poleroviruses. We examined the virus transmission characteristics of several distinct populations and various developmental stages of the aphid. Seven well-characterized S. graminum biotypes maintained at the USDA-ARS laboratory in Stillwater, OK, and two biotypes maintained in New York (one collected in Wisconsin and the other collected in South Carolina) were tested for their ability to transmit five viruses that cause barley yellow dwarf disease (BYD). Four of the Oklahoma biotypes, which do not commonly colonize agronomic crops, and the Wisconsin biotype, were efficient vectors of several viruses. The three other Oklahoma biotypes, which do colonize agronomic crops, and the South Carolina biotype, were poor vectors of all five viruses. Thus, the vector specificity long associated with viruses causing BYD is not limited to the level of aphid species; it clearly extends to populations within a single species. S. graminum nymphs are reported to be more efficient vectors of Barley yellow dwarf virus (BYDV-SGV) than are adults. This was confirmed only for the Wisconsin biotype, but not for the other eight S. graminum biotypes. Thus, there does not appear to be a generalized developmentally regulated barrier to the transmission of BYDV-SGV in S. graminum. Furthermore, the developmentally regulated vector competency observed in the Wisconsin biotype did not extend to other viruses. BYDV-PAV and Cereal yellow dwarf virus-RPV were transmitted with similar efficiency by all S. graminum biotypes when acquired by nymphs or adults.

Physiological modification of the host feeding site by cereal aphids (Homoptera: Aphididae).

Indole-3-acetic acid-l-14C and 14C-sucrose labels were used to study the effects of greenbugs, Schizaphis graminum (Rondani), and Russian wheat aphids, Diuraphis noxia (Mordvilko), on phloem function of wheat (Triticum aesticum L.). Greenbug feeding significantly reduced translocation from the immediate feeding site; however, phloem integrity was not impeded. In contrast, Russian wheat aphids had little effect on vein loading or phloem translocation at the feeding site. Similar results were obtained when resistant and susceptible wheats were infested with three different greenbug biotypes. Greenbugs fed artificial diets containing 14C-sucrose injected salivary material that was translocated to both root and shoot systems. The accumulation of salivary constituents in the roots of wheat seedlings fed upon by greenbugs may account for the significant reductions in root biomass that have previously been reported.

Measurement of Lens Autofluorescence Can Distinguish Subjects With Diabetes From Those Without.

Lens autofluorescence is increased in patients with diabetes mellitus, but clinical application has been limited by the lack of an instrument suitable for routine clinical use. We investigate possible uses of a new scanning confocal biomicroscope (1) to identify subjects with undiagnosed type 2 diabetes and (2) as a marker for the progression of diabetes. One hundred seventy-eight subjects self-reported as normal and 53 subjects physician-diagnosed with diabetes or prediabetes were recruited. Measurements were collected using a ClearPath DS-120 Lens Fluorescence Biomicroscope calibrated with standards traceable to National Institute of Standards and Technology (NIST). Fluorescence intensities were corrected for age by subtracting the value expected from a regression of intensity versus age for normal subjects. This "fluorescence deviation" showed progressively higher values for normal, prediabetes, type 2 diabetes, and type 1 diabetes and a high degree of predictability of diabetes diagnosis. A receiver operating characteristics curve was used to determine sensitivity and specificity for prediction of diabetes type 2. At a fluorescence deviation of 2500, a sensitivity of 67% at 94% specificity was observed detection of type 2 diabetes. The progressively higher fluorescence deviations are consistent with the physiological mechanisms of accumulation of fluorescent advanced glycation end products as the subject ages. The sensitivity and specificity performance of the lens autofluorescence test for type 2 diabetes is comparable to the performance of glucose threshold tests. The statistically significant difference between fluorescence deviations of normal and type 2 diabetes supports the feasibility of lens autofluorescence to screen subjects for undiagnosed type 2 diabetes. Ophthalmic practices are points of care at which there may be a public health benefit for screening patients for undiagnosed diabetes.

Simultaneous noninvasive clinical measurement of lens autofluorescence and rayleigh scattering using a fluorescence biomicroscope.

BACKGROUND: Lens autofluorescence increases with the age of the subject, and the fluorophores responsible are associated with cataract, retinopathy, and other complications of diabetes. We built a scanning confocal lens fluorescence biomicroscope suitable for routine clinical measurement of lens autofluorescence and light scattering and report data from 127 healthy subjects. METHOD: The fluorescence biomicroscope focuses a beam of light from a blue light-emitting diode on the lens and measures fluorescent green light and blue scattered light using a sensitive silicon photomultiplier. The system includes a target fixation light and a video camera for alignment and automatic pupil tracking. Under software control, a volume of measurement is scanned from behind the posterior lens capsule, through the lens to the aqueous humor, and then back again. Software computes the average ratio of lens autofluorescence to scattered light in the central portion of the lens. Self-reported healthy nondiabetic subjects were examined by an optometrist; if their eyes were healthy and without significant cataract, they were entered into the study. RESULTS: Valid lens autofluorescence data were collected from 127 subjects between 21 and 70 years of age. A linear model for lens autofluorescence intensity with age was highly statistically significant, and the improvement in fit for higher-order polynomial models was not statistically significant. The ratio of lens autofluorescence to light scatter was also calculated; regression analysis showed significant curvature for the relationship of the fluorescence ratio to age, so a nonlinear model was used to estimate the mean ratio of autofluorescence to scatter and its prediction intervals as a function of age. CONCLUSIONS: Our observation of a strongly significant linear regression of fluorescence intensity with age of the subjects agrees with the results from previous studies, as does a nonlinear model for the fluorescence ratio. The fluorescence biomicroscope enables the clinician to identify patients with fluorescence ratio significantly higher than expected for their age.