Fluorescent d-Amino Acids for Super-resolution Microscopy of the Bacterial Cell Wall.

Fluorescent d-amino acids (FDAAs) have previously been developed to enable in situ highlighting of locations of bacterial cell wall growth. Most bacterial cells lie at the edge of the diffraction limit of visible light; thus, resolving the precise details of peptidoglycan (PG) biosynthesis requires super-resolution microscopy after probe incorporation. Single molecule localization microscopy (SMLM) has stringent requirements on the fluorophore photophysical properties and therefore has remained challenging in this context. Here, we report the synthesis and characterization of new FDAAs compatible with one-step labeling and SMLM imaging. We demonstrate the incorporation of our probes and their utility for visualizing PG at the nanoscale in Gram-negative, Gram-positive, and mycobacteria species. This improved FDAA toolkit will endow researchers with a nanoscale perspective on the spatial distribution of PG biosynthesis for a broad range of bacterial species.

Alfalfa leaf curl virus is efficiently acquired by its aphid vector Aphis craccivora but inefficiently transmitted.

Alfalfa leaf curl virus (ALCV) is the first geminivirus for which aphid transmission was reported. Transmission by Aphis craccivora was determined previously to be highly specific and circulative. Using various complementary techniques, the transmission journey of ALCV was monitored from its uptake from infected plant tissues up to the head of its vector. ALCV was shown to be restricted to phloem tissues using fluorescence in situ hybridization (FISH) and electropenetrography (EPG) monitoring of virus acquisition. Furthermore, the virus is heterogeneously distributed in phloem tissues, as revealed by FISH and quantitative PCR of viral DNA acquired by EPG-monitored aphids. Despite the efficient ingestion of viral DNA, about 106 viral DNA copies per insect in a 15 h feeding period on ALCV-infected plants, the individual maximum transmission rate was 12 %. Transmission success was related to a critical viral accumulation, around 1.6×107 viral DNA copies per insect, a threshold that generally needed more than 48 h to be reached. Moreover, whereas the amount of acquired virus did not decrease over time in the whole aphid body, it declined in the haemolymph and heads. ALCV was not detected in progenies of viruliferous aphids and did not affect aphid fitness. Compared to geminiviruses transmitted by whiteflies or leafhoppers, or to luteoviruses transmitted by aphids, the transmission efficiency of ALCV by A. craccivora is low. This result is discussed in relation to the aphid vector of this geminivirus and the agroecological features of alfalfa, a hardy perennial host plant.

Co-Acquired Nanovirus and Geminivirus Exhibit a Contrasted Localization within Their Common Aphid Vector.

Single-stranded DNA (ssDNA) plant viruses belong to the families Geminiviridae and Nanoviridae. They are transmitted by Hemipteran insects in a circulative, mostly non-propagative, manner. While geminiviruses are transmitted by leafhoppers, treehoppers, whiteflies and aphids, nanoviruses are transmitted exclusively by aphids. Circulative transmission involves complex virus-vector interactions in which epithelial cells have to be crossed and defense mechanisms counteracted. Vector taxa are considered a relevant taxonomic criterion for virus classification, indicating that viruses can evolve specific interactions with their vectors. Thus, we predicted that, although nanoviruses and geminiviruses represent related viral families, they have evolved distinct interactions with their vector. This prediction is also supported by the non-structural Nuclear Shuttle Protein (NSP) that is involved in vector transmission in nanoviruses but has no similar function in geminiviruses. Thanks to the recent discovery of aphid-transmitted geminiviruses, this prediction could be tested for the geminivirus alfalfa leaf curl virus (ALCV) and the nanovirus faba bean necrotic stunt virus (FBNSV) in their common vector, Aphis craccivora. Estimations of viral load in midgut and head of aphids, precise localization of viral DNA in cells of insect vectors and host plants, and virus transmission tests revealed that the pathway of the two viruses across the body of their common vector differs both quantitatively and qualitatively.

Infection of a French Population of Aedes albopictus and of Aedes aegypti (Paea Strain) with Zika Virus Reveals Low Transmission Rates to These Vectors' Saliva.

Disease caused by the Zika virus (ZIKV) is a public health emergency of international concern. Recent epidemics have emerged in different regions of the world and attest to the ability of the virus to spread wherever its vector, Aedes species mosquitoes, can be found. We have compared the transmission of ZIKV by Ae. aegypti (PAEA strain originating from Tahiti) and by a French population of Ae. albopictus to better assess their competence and the potential risk of the emergence of ZIKV in Europe. We assessed the transmission of ZIKV by Ae. albopictus in temperatures similar to those in Southern France during the summer. Our study shows that the extrinsic incubation period of Ae. aegypti for transmission was shorter than that of Ae. albopictus. Both vectors were able to transmit ZIKV from 10 to 14 days post-infection. Ae. aegypti, however, had a longer transmission period than the French population of Ae. albopictus. Although the salivary glands of both vectors are highly infected, transmission rates of ZIKV to saliva remain relatively low. These observations may suggest that the risk of emergence of ZIKV in Europe could be low.