Laser microdissection of grapevine leaf phloem infected by stolbur reveals site‐specific gene responses associated to sucrose transport and metabolism

Bois Noir is an emergent disease of grapevine that has been associated to a phytoplasma belonging to the XII‐A stolbur group. In plants, phytoplasmas have been found mainly in phloem sieve elements, from where they spread moving through the pores of plates, accumulating especially in source leaves. To examine the expression of grapevine genes involved in sucrose transport and metabolism, phloem tissue, including sieve element/companion cell complexes and some parenchyma cells, was isolated from healthy and infected leaves by means of laser microdissection pressure catapulting (LMPC). Site‐specific expression analysis dramatically increased sensitivity, allowing us to identify specific process components almost completely masked in whole‐leaf analysis. Our findings showed decreased phloem loading through inhibition of sucrose transport and increased sucrose cleavage activity, which are metabolic changes strongly suggesting the establishment of a phytoplasma‐induced switch from carbohydrate source to sink. The analysis focused at the infection site also showed a differential regulation and specificity of two pathogenesis‐related thaumatin‐like genes (TL4 and TL5) of the PR‐5 family.     

Integrating vibrational signals,mitochondrial DNA and morphology for species determination in the genus Aphrodes (Hemiptera: Cicadellidae)

Reliable delimitation and identification of species is central not only to systematics, but also to studies of biodiversity, ecology and pest management. In the era of Internet‐based biodiversity databases misidentifications are rapidly disseminated and may have far‐reaching consequences. Leafhoppers from the genus Aphrodes (Hemiptera, Cicadellidae) are common and abundant, but, nevertheless, they are still a taxonomically challenging group whose members are often assessed in ecological studies and are also potential vectors of plant diseases. Previous study has shown that the syntype series for A. aestuarina (Edwards) includes also specimens of A. makarovi Zachvatkin and has suggested that misidentifications may be widespread in museum collections. We studied Aphrodes individuals collected from the U.K. and Slovenia in order to provide a more comprehensive analysis of this genus using multiple criteria. Combined work using male and female vibrational signals emitted during courtship, and a 600‐bp fragment within the barcoding region of the COI mtDNA gene, provided validated specimens that we also used for morphometric study. Analyses confirmed A. aestuarina, A. bicincta, A. diminuta and A. makarovi as behaviourally, genetically and morphologically distinct species. Although any of these approaches could be used alone to distinguish between species, combining morphological and molecular approaches will help to improve reliability, especially when identifying females. Morphological investigation of validated individuals from the U.K. and Slovenia also revealed geographic differences within species. By combining several body and aedeagus morphological characters males can be reliably identified, however, morphological differences between species are, nevertheless, relatively small. By contrast, observed genetic distances between Aphrodes species are relatively large (4.2–7.0%). At about half of our collecting sites more than one Aphrodes species was found and A. makarovi was collected together with every other species, including A. aestuarina on tidal saltmarshes. Due to low morphological variation between syntopic congeners it is likely that many museum specimens of Aphrodes have been assigned to the wrong species and species identification in ecological and vector studies may also be questionable.     

Dermal Bioavailability of Toluene Aged in Soil

When organic chemicals age in soil, they can become more sequestered with time and less bioavailable. The impact of aging in soil was assessed on the dermal bioavailability of toluene. Comparisons were made between the dermal bioavailability of toluene aged in two soils (Atsion and Keyport) for 3 months, toluene in freshly spiked soil, and toluene without soil (pure chemical). In vitro flow-through diffusion cell methodology measured the amount of radioactive chemical that penetrated dermatomed male pig skin into receptor fluid, and that was detected in skin following soap and water decontamination. Volatilization reduced the amount of toluene that was available for skin penetration. Therefore, relative to the available dose, the total penetration of pure toluene (sum of the dose in the receptor fluid and skin) was 93%. The majority of pure toluene was found in skin (82%). The dermal bioavailability of non-aged and aged toluene was decreased to 4–6% and 3–4% of the available dose, respectively. The data suggest that there would be a lower potential risk to human health from dermal exposure to toluene in soil. However, lower soil loading and longer aging time are needed to quantitate the risk.     

ACROSOME BREAKDOWN IN LEPTODACTYLUS CHAQUENSIS (AMPHIBIA ANURA) SPERMATOZOA*

Acrosome breakdown in Leptodactylus chaquensis is described: during this process acrosome enlarges, becomes round-shaped and finally disrupts. Low tonicity media (0.025 M sucrose and 1/10 Holtfreter's solutions) favor acrosome breakdown and sperm fertility loosing. High tonicity media (0.250 M sucrose and Holtfreter's solutions) maintain acrosomes in an unreacted stage and sperm fertilizing capacity is preserved. Sperm motility does not seem to be a sufficient condition for the sperm to fertilize and also does not seem to be related with acrosome breakdown. The presence of lectins in the incubation media does not modify the time-course of acrosome breakdown.