Secreted microRNA data from the parasitic filarial nematode Acanthocheilonema viteae

microRNAs (miRNAs) are an abundant class of non-coding RNA species with important regulatory roles in gene expression at the posttranscriptional level. The helminth; Acanthocheilonema viteae; serves as model organism for research on parasitic filarial nematodes. Total RNA secreted or excreted; in vitro; by 1500 adult female and male; A. viteae; over 3 weeks was isolated from culture media previously processed by differential ultracentrifugation. miRNA sequencing revealed the presence of 360 unique miRNA candidates released by adult; A. viteae in vitro; . Among them, 74 high-confidence unique miRNAs; ,; as well as several potential novel miRNA candidates were discovered. A large proportion of the sequenced miRNA candidates appeared differentially expressed between the male and female samples based on normalized copy count. The presence of extracellular vesicles, often rich in miRNAs, could not be confirmed unambiguously by transmission electron microscopy

Nematode microRNAs can Individually Regulate Interferon Regulatory Factor 4 and mTOR in Differentiating T Helper 2 Lymphocytes and Modulate Cytokine Production in Macrophages

Parasitic nematodes are masterful immunomodulators. This class of pathogens has evolved a spectrum of sophisticated strategies to regulate and evade host immune responses, mediated through the release of various molecules. In this context, the release of microRNAs (miRNAs), short post-transcriptional regulators of gene expression, has been of particular interest in the host-parasite interplay. Evidence that parasite-derived miRNAs modulate host innate and adaptive immune responses has become increasingly compelling. However, since miRNAs are usually contained in extracellular vesicles containing other mediators, it is difficult to assign an observed effect on host cells to miRNAs specifically. Here, the effects of some abundantly secreted miRNAs by nematodes used as models of gastrointestinal infections (Heligmosomoides polygyrus bakeri, Trichuris muris and Ascaris suum) were evaluated, addressing the potential of parasite miRNAs to impair in vitro differentiation of two important types of immune cells in the context of helminth infections, Th2 lymphocytes and macrophages. Mimicking a continuous exposure to low concentrations of nematode miRNAs, the interferon gamma signaling, the IL-2/STAT5 signaling, and the mTOR signaling pathways were identified as downregulated by Hpo-miR-71-5p. Interferon regulatory factor 4 (Irf4) was validated as a target of Hpo-miR-71-5p, while Mtor is targeted by Asu-miR-791-3p, abundant in the T. muris secretions. By trend, Hpo-miR-71-5p impacts mildly but consistently on the amounts of inflammatory cytokines in unpolarized macrophages but leads to slightly increased IL-10 level in alternatively activated cells. In addition, our data suggests that transfected miRNAs remain for days in recipient cells, and that Hpo-miR-71-5p can incorporate into mouse Argonaute protein complexes. Nematode miRNAs can impair both innate and adaptive arms of host immunity. Hpo-miR-71-5p in particular, absent in mammals, interacts with host genes and pathways with crucial involvement in anthelmintic immune responses. This report brings new insights into the dynamics of miRNA-driven immunomodulation and highlights putative targeted pathways. Although the absolute repression is subtle, it is expected that the dozens of different miRNAs released by nematodes may have a synergistic effect on surrounding host cells

High-throughput Sorting of Mosquito Larvae for Laboratory Studies and for Future Vector Control Interventions

Background: Mosquito transgenesis offers new promises for the genetic control of vector-borne infectious diseases such as malaria and dengue fever. Genetic control strategies require the release of large number of male mosquitoes into field populations, whether they are based on the use of sterile males (sterile insect technique, SIT) or on introducing genetic traits conferring refractoriness to disease transmission (population replacement). However, the current absence of high-throughput techniques for sorting different mosquito populations impairs the application of these control measures. Methods: A method was developed to generate large mosquito populations of the desired sex and genotype. This method combines flow cytometry and the use of Anopheles gambiae transgenic lines that differentially express fluorescent markers in males and females. Results: Fluorescence-assisted sorting allowed single-step isolation of homozygous transgenic mosquitoes from a mixed population. This method was also used to select wild-type males only with high efficiency and accuracy, a highly desirable tool for genetic control strategies where the release of transgenic individuals may be problematic. Importantly, sorted males showed normal mating ability compared to their unsorted brothers. Conclusions: The developed method will greatly facilitate both laboratory studies of mosquito vectorial capacity requiring high-throughput approaches and future field interventions in the fight against infectious disease vectors

Transgenic Expression of the Anti-parasitic Factor TEP1 in the Malaria Mosquito Anopheles gambiae

Mosquitoes genetically engineered to be resistant to Plasmodium parasites represent a promising novel approach in the fight against malaria. The insect immune system itself is a source of anti-parasitic genes potentially exploitable for transgenic designs. The Anopheles gambiae thioester containing protein 1 (TEP1) is a potent anti-parasitic protein. TEP1 is secreted and circulates in the mosquito hemolymph, where its activated cleaved form binds and eliminates malaria parasites. Here we investigated whether TEP1 can be used to create malaria resistant mosquitoes. Using a GFP reporter transgene, we determined that the fat body is the main site of TEP1 expression. We generated transgenic mosquitoes that express TEP1r, a potent refractory allele of TEP1, in the fat body and examined the activity of the transgenic protein in wild-type or TEP1 mutant genetic backgrounds. Transgenic TEP1r rescued loss-of-function mutations, but did not increase parasite resistance in the presence of a wild-type susceptible allele. Consistent with previous reports, TEP1 protein expressed from the transgene in the fat body was taken up by hemocytes upon a challenge with injected bacteria. Furthermore, although maturation of transgenic TEP1 into the cleaved form was impaired in one of the TEP1 mutant lines, it was still sufficient to reduce parasite numbers and induce parasite melanization. We also report here the first use of Transcription Activator Like Effectors (TALEs) in Anopheles gambiae to stimulate expression of endogenous TEP1. We found that artificial elevation of TEP1 expression remains moderate in vivo and that enhancement of endogenous TEP1 expression did not result in increased resistance to Plasmodium. Taken together, our results reveal the difficulty of artificially influencing TEP1-mediated Plasmodium resistance, and contribute to further our understanding of the molecular mechanisms underlying mosquito resistance to Plasmodium parasites

Targeted Mutagenesis in the Malaria Mosquito Using TALE Nucleases

Anopheles gambiae, the main mosquito vector of human malaria, is a challenging organism to manipulate genetically. As a consequence, reverse genetics studies in this disease vector have been largely limited to RNA interference experiments. Here, we report the targeted disruption of the immunity gene TEP1 using transgenic expression of Transcription-Activator Like Effector Nucleases (TALENs), and the isolation of several TEP1 mutant A. gambiae lines. These mutations inhibited protein production and rendered TEP1 mutants hypersusceptible to Plasmodium berghei. The TALEN technology opens up new avenues for genetic analysis in this disease vector and may offer novel biotechnology-based approaches for malaria control

Transgenic Expression of the Anti-parasitic Factor TEP1 in the Malaria Mosquito Anopheles gambiae

Mosquitoes genetically engineered to be resistant to Plasmodium parasites represent a promising novel approach in the fight against malaria. The insect immune system itself is a source of anti-parasitic genes potentially exploitable for transgenic designs. The Anopheles gambiae thioester containing protein 1 (TEP1) is a potent anti-parasitic protein. TEP1 is secreted and circulates in the mosquito hemolymph, where its activated cleaved form binds and eliminates malaria parasites. Here we investigated whether TEP1 can be used to create malaria resistant mosquitoes. Using a GFP reporter transgene, we determined that the fat body is the main site of TEP1 expression. We generated transgenic mosquitoes that express TEP1r, a potent refractory allele of TEP1, in the fat body and examined the activity of the transgenic protein in wild-type or TEP1 mutant genetic backgrounds. Transgenic TEP1r rescued loss-of-function mutations, but did not increase parasite resistance in the presence of a wild-type susceptible allele. Consistent with previous reports, TEP1 protein expressed from the transgene in the fat body was taken up by hemocytes upon a challenge with injected bacteria. Furthermore, although maturation of transgenic TEP1 into the cleaved form was impaired in one of the TEP1 mutant lines, it was still sufficient to reduce parasite numbers and induce parasite melanization. We also report here the first use of Transcription Activator Like Effectors (TALEs) in Anopheles gambiae to stimulate expression of endogenous TEP1. We found that artificial elevation of TEP1 expression remains moderate in vivo and that enhancement of endogenous TEP1 expression did not result in increased resistance to Plasmodium. Taken together, our results reveal the difficulty of artificially influencing TEP1-mediated Plasmodium resistance, and contribute to further our understanding of the molecular mechanisms underlying mosquito resistance to Plasmodium parasites

Salivary gland-specific P. berghei reporter lines enable rapid evaluation of tissue-specific sporozoite loads in mosquitoes

Malaria is a life-threatening human infectious disease transmitted by mosquitoes. Levels of the salivary gland sporozoites (sgs), the only mosquito stage infectious to a mammalian host, represent an important cumulative index of Plasmodium development within a mosquito. However, current techniques of sgs quantification are laborious and imprecise. Here, transgenic P. berghei reporter lines that produce the green fluorescent protein fused to luciferase (GFP-LUC) specifically in sgs were generated, verified and characterised. Fluorescence microscopy confirmed the sgs stage specificity of expression of the reporter gene. The luciferase activity of the reporter lines was then exploited to establish a simple and fast biochemical assay to evaluate sgs loads in whole mosquitoes. Using this assay we successfully identified differences in sgs loads in mosquitoes silenced for genes that display opposing effects on P. berghei ookinete/oocyst development. It offers a new powerful tool to study infectivity of P. berghei to the mosquito, including analysis of vector-parasite interactions and evaluation of transmission-blocking vaccines

Coléoptères de la ville de Dijon et de sa périphérie urbaine (Côte-d'Or) -Troisième partie

Prost Monique, Soichot Julien. Coléoptères de la ville de Dijon et de sa périphérie urbaine (Côte-d'Or) -Troisième partie. In: Bulletin mensuel de la Société linnéenne de Lyon, 79ᵉ année, n°7-8, Septembre-octobre 2010. pp. 209-240

Coléoptères de la ville de Dijon et de sa périphérie urbaine (Côte-d'Or) - Première partie

Coleopterous insects from the city of Dijon and its outskirts (Côte-d'Or, Burgundy) -First part. Too often neglected by the entomologists, cities are though sheltering several insects species. Green areas (parks, gardens, trees alignments, etc.) probably play a major role in maintaining this biodiversity. Dijon is a city with many gardens, public parks, and vast periurban parks and appears as an ideal place to study insects. Jean Barbier (1913-1980) captured many Coleoptera species there between 1938 and 1980. From his collection, completed with the main entomological collection of the Museum of Dijon and datas provided by the members of the Dijon Entomological Society, we established a catalogue of Coleoptera captured in Dijon agglomeration since the end of the XIXth century. We made a census of 1150 species.La plupart du temps délaissées par les entomologistes, les villes abritent pourtant de nombreuses espèces d'insectes. Les espaces verts (parcs, jardins, alignements d'arbres, etc.) jouent un rôle essentiel dans le maintien d'une entomofaune plus riche en espèces qu'on ne le soupçonne habituellement. L'agglomération dijonnaise avec ses jardins et ses parcs, en particulier le Bois du Parc de la Colombière, le plus grand espace vert au cœur même de la ville de Dijon, avec ses combes périurbaines, offre un terrain d'étude remarquable. Jean Barbier (1913-1 980) y a récolté de nombreuses espèces de Coléoptères entre 1938 et 1980. À partir de sa collection, complétée par la collection générale du Muséum-Jardin des Sciences de la ville de Dijon et par les données recueillies par les membres de la Société Entomologique de Dijon, en particulier pour les Cerambycidae qu'il avait peu étudiés, nous avons dressé un catalogue des Coléoptères capturés depuis la fin du XIXe siècle jusqu'à nos jours dans toute l'agglomération dijonnaise. Nous y avons recensé 1150 espèces.Prost Monique, Soichot Julien. Coléoptères de la ville de Dijon et de sa périphérie urbaine (Côte-d'Or) - Première partie. In: Bulletin mensuel de la Société linnéenne de Lyon, 79ᵉ année, n°3-4, Mars-avril 2010. pp. 47-82

Coléoptères de la ville de Dijon et de sa périphérie urbaine (Côte-d'Or) -Deuxième partie

Prost Monique, Soichot Julien. Coléoptères de la ville de Dijon et de sa périphérie urbaine (Côte-d'Or) -Deuxième partie. In: Bulletin mensuel de la Société linnéenne de Lyon, 79ᵉ année, n°5-6, Mai-juin 2010. pp. 119-166