RNAi pathway genes are not modulated by VSV infection in <i>L</i>. <i>longipalpis</i>.

<p><b>(A)</b> Expression of <i>L</i>. <i>longipalpis</i> genes encoding <i>Dicer-2</i>, <i>AGO2</i> and <i>r2d2</i> in control and VSV-infected LL5 cells at different time points. <b>(B)</b> Expression of <i>L</i>. <i>longipalpis</i> genes encoding <i>Dicer-2, AGO2 and r2d2</i> in adult sandflies fed with a blood meal containing VSV. Control sandflies (Mock) were fed with blood without virus. Black circles indicate individuals with detectable viral RNA levels. Numbers of infected individuals and the total are indicated at each time point. No significant differences were observed.</p

VSV replication in adult <i>L</i>. <i>longipalpis</i>.

<p><b>(A)</b> Adult <i>L</i>. <i>longipalpis</i> sandflies were fed with blood containing different concentrations of VSV and the prevalence of infection was analyzed by RT-qPCR at 2 and 4 dpf. The number of individuals tested is indicated at each time point. <b>(B)</b> Adult sandflies were given a blood meal containing 10⁸ PFU/mL of VSV and viral RNA levels in individual sandflies were determined at 1, 2, 4 and 6 dpf. The number of infected individuals is indicated at each time point. Control sandflies (Mock) were fed with blood without virus. Black circles indicate individuals with detectable viral RNA levels. Individuals used to prepare small RNA libraries shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006569#pntd.0006569.g003" target="_blank">Fig 3</a> are indicated in green. Scatter dot plot shows median with interquatile ranges. Statistical significance was determined using Dunn’s Multiple Comparison test for infected groups at different times after feeding. Significant <i>p</i> values are indicated in the figure. <b>(C)</b> <i>L</i>. <i>longipalpis</i> were given a blood meal with containing 10⁸ PFU/mL of VSV and infectious particles in each individual were assayed by PFU. The number of infected individuals and the total are indicated at each time point. Black circles indicate individuals with detectable infectious particles. Experiments are representative of at least 3 biological replicates.</p

The small non-coding RNA response to virus infection in the <i>Leishmania</i> vector <i>Lutzomyia longipalpis</i>

<div><p>Sandflies are well known vectors for <i>Leishmania</i> but also transmit a number of arthropod-borne viruses (arboviruses). Few studies have addressed the interaction between sandflies and arboviruses. RNA interference (RNAi) mechanisms utilize small non-coding RNAs to regulate different aspects of host-pathogen interactions. The small interfering RNA (siRNA) pathway is a broad antiviral mechanism in insects. In addition, at least in mosquitoes, another RNAi mechanism mediated by PIWI interacting RNAs (piRNAs) is activated by viral infection. Finally, endogenous microRNAs (miRNA) may also regulate host immune responses. Here, we analyzed the small non-coding RNA response to <i>Vesicular stomatitis virus</i> (VSV) infection in the sandfly <i>Lutzoymia longipalpis</i>. We detected abundant production of virus-derived siRNAs after VSV infection in adult sandflies. However, there was no production of virus-derived piRNAs and only mild changes in the expression of vector miRNAs in response to infection. We also observed abundant production of virus-derived siRNAs against two other viruses in <i>Lutzomyia</i> Lulo cells. Together, our results suggest that the siRNA but not the piRNA pathway mediates an antiviral response in sandflies. In agreement with this hypothesis, pre-treatment of cells with dsRNA against VSV was able to inhibit viral replication while knock-down of the central siRNA component, Argonaute-2, led to increased virus levels. Our work begins to elucidate the role of RNAi mechanisms in the interaction between <i>L</i>. <i>longipalpis</i> and viruses and should also open the way for studies with other sandfly-borne pathogens.</p></div

Production of virus-derived small RNAs in <i>L</i>. <i>longipalpis</i>.

<p><b>(A)</b> Analysis of small RNA libraries from control <i>L</i>. <i>longipalpis</i> (mock) at 2, 4 and 6 dpf shows absence of VSV-derived small RNAs. <b>(B)</b> Size distribution of VSV-derived small RNAs at 2, 4 and 6 dpf in infected <i>L</i>. <i>longipalpis</i> shows a symmetrical peak at 21 nt that is characteristic of siRNAs. <b>(C)</b> Coverage of virus-derived small RNAs along the VSV genome from libraries prepared at 2, 4 and 6 dpf shows homogenous and symmetrical distribution. <b>(D)</b> Size distribution of small RNAs derived from LV1 and LV2 found in Lulo cells derived from <i>Lutzomyia</i> shows a symmetrical peak at 21 nt that is characteristic of siRNAs. <b>(E)</b> Coverage of virus-derived small RNAs along the genome of LV1 and LV2 from libraries prepared from Lulo cells shows homogenous and symmetrical distribution. 5’ base preferences of small RNAs are indicated by color. Distribution of small RNAs over sense (blue) and anti-sense (brown) strands of the viral genomes are indicated.</p

VSV replication in <i>Lutzomyia</i> cell lines.

<p><b>(A)</b> Kinetics of VSV replication in LL5 cell using a MOI of 10 PFU/mL. Statistical significance was determined using Tukey’s Multiple Comparison test between infected groups at different times post infection. This experiment is representative of three biological replicates. Significant p values are indicated in the figure. <b>(B)</b> Infectious VSV particles produced in culture supernatants of LL5 cells were assayed by PFU in Vero cell. This experiment is representative of three biological replicates <b>(C)</b> Analysis of cell monolayers at different times after VSV infection in Vero, C636 and LL5 cells. <b>(D)</b> Infectious viral particles produced in the supernatant at different points after VSV infection in Vero, C636 and LL5 cells with a MOI of 0.1 PFU/mL. Experiments are representative of at least two biological replicates.</p

Absence of virus-derived piRNAs in <i>L</i>. <i>longipapis</i>.

<p><b>(A)</b> Size distribution of small RNAs derived from VSV, LV1 and LV2 in the 24 to 35 nt range considering each strand separately. 5’ base preferences are indicated by color. <b>(B)</b> Nucleotide preferences for each position of small RNAs between 24–30 nt are shown as a weblogo. The number of reads analyzed is indicated in brackets. <b>(C)</b> Relative frequency of overlap between 5’ end of small RNAs between 24–30 nt in opposite strands.</p

<i>Phlebotomus papatasi</i> circadian rhythm pathway annotation.

Phlebotomus papatasi circadian rhythm pathway annotation.</p

Chitinase family annotation.

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.</div

Molecular phylogenetic analysis of <i>Lu</i>. <i>longipalpis</i>, <i>P</i>. <i>papatasi</i> and <i>D</i>. <i>melanogaster</i> TRP channel sequences.

The different TRP subfamilies are displayed on the right. The evolutionary history was inferred by using the Maximum Likelihood method based on the Whelan and Goldman +Freq. model with 1000 bootstrap replicates. (TIF)</p

Glycosidase Hydrolase family 13 annotation.

Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.</div