Interactions of maize bushy stunt phytoplasma with the leafhopper vector, Dalbulus maidis (Delong and Wolcott) (Hemiptera: Cicadellidae) and associated microbiota

Phytoplasmas are bacteria with a persistent propagative transmission by insect vectors that generates direct and indirect interactions among them. In order to understand these interactions for maize bushy stunt phytoplasma (MBSP) and the leafhopper vector Dalbulus maidis (Hemiptera: Cicadellidae), two research lines were addressed. The first one aimed to determine the indirect effects of maize infection by MBSP on some biological and behavioral parameters of the vector, whereas a second line investigated direct interactions of the phytoplasma with D. maidis during its movement through the vector body following acquisition from plants, and associated microbiota. Indirect effects were investigated in choice experiments in which alighting and oviposition preferences by D. maidis were compared on healthy vs. MBSP-infected plants with variable incubation time (diseased plants with early and advanced symptoms, or still asymptomatic). Likewise, indirect effect of MBSP on the D. maidis biology was determined in two life table experiments in which the vector was reared on healthy vs. MBSP-infected plants expressing advanced disease symptoms or still asymptomatic. Choice experiments showed that alighting and oviposition preferences of D. maidis on MBSP-infected plants compared to healthy plants depend on the pathogen incubation period in the plant. The leafhopper preferred MBSP-infected plants over healthy ones during the asymptomatic phase of the disease, but rejected infected plants with advanced symptoms. The vector was able to acquire MBSP from asymptomatic infected plants shortly (3 days) after inoculation, but transmission efficiency increased when acquisition occurred at later stages of the pathogen incubation period (≥14 days) in the source plants and the test plants showed disease symptoms faster. These results suggest that MBSP modulates D. maidis preference for asymptomatic infected plants in the early stages of the crop, allowing rapid spread of this pathogen. Maize infection by the phytoplasma had a neutral effect on most life table parameters of D. maidis; a lower net reproductivity rate (Ro) was observed in the cohort reared on MBSP-infected plants with advanced symptoms, which was compensated to some extent by a higher sexual ratio. MBSP acquisition by all vector nymphal stadia was confirmed by PCR, and the pathogen as detected in both male and female reproductive organs. Concerning direct MBSP-vector interactions, transmission electron microscopy analyses showed phytoplasma-like cells in the midgut lumen, microvilli and epithelial cells, suggesting that MBSP enters the epithelium midgut through the microvilli wall. Within the epithelial cells, mitochondria and bacteria-like cells (possibly endosymbionts) were observed together with masses of phythoplasma-like cells. In the hemocoel, phytoplasma-like cells grouped into a matrix were also observed in association with bacteria-like cells similar to those observed in the midgut epithelium. Similar associations were found in the salivary gland. Interestingly, in-situ hybridization (FISH) technique revealed a variation in diversity and abundance of the microbiota in intestine and salivary glands of D. maidis adults over time after MBSP acquisition from plants. Sulcia sp., Cardinium sp. and eubacteria increased their abundance over time, whereas Rickettsia sp. decreased. The frequent association of the vector microbiota with the phytoplasma in some tissues of D. maidis suggests that endosymbiotic bacteria may play some role in MBSP-vector interactions.

Incubation experiments to examine the production and loss of CH3I in surface seawater

In order to investigate production pathways of methyl iodide and controls on emissions from the surface ocean, a set of repeated in-vitro incubation experiments were performed over an annual cycle in the context of a time-series of in-situ measurements in Kiel Fjord (54.3 N, 10.1E). The incubation experiments revealed a diurnal variation of methyl iodide in samples exposed to natural light, with maxima during day time and losses during night hours. The amplitude of the daily accumulation varied seasonally and was not affected by filtration (0.2µm), consistent with a photochemical pathway for CH3I production. The methyl iodide loss rate during night time correlated with the concentration accumulated during daytime. Daily (24 hour) net production (Pnet) was similar in magnitude between in vitro and in situ mass balances. However, the estimated gross production (Pgross) of methyl iodide ranged from -0.07 to 2.24 pmol/day and were 5 times higher in summer than Pnet calculated from the in-situ study [Shi et al., 2014]. The large excess of Pgross over Pnet revealed by the in-vitro (incubation) experiments in summer is a consequence of large losses of CH3I by as-yet uncharacterized processes (e.g. biological degradation or chemical pathways other than Cl- substitution).

Benthic anammox and dentrification rates measured in a slurry incubation experiments at four different stations in the Arabian Sea off Pakistan during the METEOR cruise M74/2 in 2007

Vertical distributions of benthic denitrification and anammox rates within the sediment were estimated from slurry incubation experiments. Rates were used to calculate the contribution of anammox and denitrification to the total N-loss. Briefly, MUC sediment cores were sliced in 2 cm intervals and the sediment was diluted and incubated with degassed bottom water in a gas tight bag. After pre-incubating the bags for 2 h, 15N-labeled substrates were injected into the bags and the slurries were thoroughly mixed. Incubations were performed in the dark at in situ temperatures. The N2 isotope ratio (28N2, 29N2, and 30N2) was determined by gas chromatography-isotopic ratio mass spectrometry (VG Optima, Micromass) and calculated according to Kuypers et al. (2005) and Holtappels et al. (2011), respectively.Furthermore, total organic carbon and nitrogen concentrations were measured of core sediment layers corresponding to those used for rate measurements. Concentrations of organic carbon and nitrogen were determined by combustion/gas chromatography (Carlo Erba NA-1500 CNS analyzer) of dried sediment samples after acidification. The same sediment layer were also used to extract nucleic acids. The concentrations of the DNA in the samples were measured spectrophotometrically with a NanoDrop instrument (Thermo Fisher Scientific Inc.). The biomarker functional gene nirS, encoding the cd1-containing nitrite reductase, for both denitrifiers and marine anammox bacteria were quantified with real-time PCR, using the primers cd3aF/R3cd (5'-GTSAACGTSAAGGARACSGG-3' (Michotey et al., 2000)/5'-GASTTCGGRTGSGTCTTGA-3'; Throback et al., 2004) and Scnir372F/Scnir845R (5'-TGTAGCCAGCATTGTAGCGT-3'/5'-TCAAGCCAGACCCATTTGCT-3'; Lam et al., 2009).

An inquiry how to prevent the small-pox and proceedings of a society for promoting general inoculation at stated periods, and preventing the natural small-pox in Chester /

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Observations medical and political on the small-pox and the advantages and disadvantages of general inoculation especially in cities, and on the mortality of mankind at every age in city and country... /

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The practice and theory of inoculation : : with an account if its success in a letter to a friend /

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An account of the Preparation and Management necessary to inoculation /

Las [4] p. finales contienen: "Books, printed for Paul Vaillant ..."

Essay upon the effects of camphire and calomel in continual fevers : : illustrated by several cases to which is added an occasional observation upon the modern practise of inoculation /

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The analysis of inoculation : comprizing the history, theory and practice of it ... /

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