List of known miRNAs identified in <i>An. stephensi</i>.

<p>aga  =  <i>Anopheles gambiae</i>; aae  =  <i>Aedes aegypti</i>; cqu  =  <i>Culex</i>; dme  =  <i>Drosophila melanogaster</i>; bmo  =  <i>Bombyx mori</i>; ame  =  <i>Apis mellifera</i>; api =  <i>Acyrthosiphon pisum</i>; tca  =  <i>Tribolium castaneum</i>.</p><p>*miRNAs present in all eight insect species.</p><p>•miRNAs not previously identified in mosquitoes.</p

Blood Feeding and <i>Plasmodium</i> Infection Alters the miRNome of <i>Anopheles stephensi</i>

<div><p>Blood feeding is an integral process required for physiological functions and propagation of the malaria vector <i>Anopheles</i>. During blood feeding, presence of the malaria parasite, <i>Plasmodium</i> in the blood induces several host effector molecules including microRNAs which play important roles in the development and maturation of the parasite within the mosquito. The present study was undertaken to elucidate the dynamic expression of miRNAs during gonotrophic cycle and parasite development in <i>Anopheles stephensi</i>. Using next generation sequencing technology, we identified 126 miRNAs of which 17 were novel miRNAs. The miRNAs were further validated by northern hybridization and cloning. Blood feeding and parasitized blood feeding in the mosquitoes revealed regulation of 13 and 16 miRNAs respectively. Expression profiling of these miRNAs revealed that significant miRNAs were down-regulated upon parasitized blood feeding with a repertoire of miRNAs showing stage specific up-regulation. Expression profiles of significantly modulated miRNAs were further validated by real time PCR. Target prediction of regulated miRNAs revealed overlapping targeting by different miRNAs. These targets included several metabolic pathways including metabolic, redox homeostasis and protein processing machinery components. Our analysis revealed tight regulation of specific miRNAs post blood feeding and parasite infection in <i>An. stephensi</i>. Such regulated expression suggests possible role of these miRNAs during gonotrophic cycle in mosquito. Another set of miRNAs were also significantly regulated at 42 h and 5 days post infection indicating parasite stage-specific role of host miRNAs. This study will result in better understanding of the role of miRNAs during gonotrophic cycle and parasite development in mosquito and can probably facilitate in devising novel malaria control strategies at vector level.</p></div

Composition of small RNA in sugar fed naive female mosquitoes 6–8 days old (SF), blood fed female mosquitoes at 42 hours (BF 42 h), 5 days post blood feeding (BF 5d), female mosquitoes at 42 hours (iBF 42 h) and 5 days post infected blood feeding (iBF 5d).

<p>Composition of small RNA in sugar fed naive female mosquitoes 6–8 days old (SF), blood fed female mosquitoes at 42 hours (BF 42 h), 5 days post blood feeding (BF 5d), female mosquitoes at 42 hours (iBF 42 h) and 5 days post infected blood feeding (iBF 5d).</p

KOBAS analysis of miRNA targets predicted by RNA hybrid.

<p>Pie charts represents significant pathways (P value<0.05) targeted by miRNAs regulated at (<b>A</b>) BF42 h, (<b>B</b>) iBF 42 h and (<b>C</b>) iBF 5d respectively. Pie chart area for each pathway represents percentage of regulated miRNAs targeting it. miRNAs are listed in the pie area of respective pathway it is targeting. Pathway marked star are targeted by maximum number of miRNAs.</p

List of novel miRNAs identified in <i>An. stephensi</i>.

<p>¥ novel miRNAs validated by Northern hybridization. miRNA ast-nv-4-5p was validated using sample SF; miRNA ast-nv-3-5p was validated using sample BF 42 h.</p

miRNA-mRNA interaction network.

<p>Interaction network of regulated miRNAs and their targets at (<b>A</b>) BF 42 h, (<b>B</b>) iBF 42 h and (<b>C</b>) iBF 5d. miRNAs are diamond shaped whereas targets are circular. Up-regulated miRNAs are highlighted in red whereas down-regulated are highlighted in green colour. Transcripts targeted by two or more miRNAs are marked in yellow colour. Significance of the Transcripts marked (*) are explained in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098402#s4" target="_blank">discussion</a>.</p

Length distribution of small RNA reads in (A) sugar fed naive female mosquitoes 6–8 days old (SF).

<p>(<b>B</b>) Female mosquitoes at 42 h post blood feeding (BF 42 h). (<b>C</b>) 5 days post blood feeding (BF 5d). (<b>D</b>) Female mosquitoes at 42 h (iBF 42 h) and (<b>E</b>) 5 days post infected blood feeding (iBF 5d). X axis represents small RNA read lengths in base pairs while Y axis represents number of reads. (<b>F</b>) Distribution of abundant and rare miRNAs in all samples. Blue and Red bars represents abundant and rare miRNAs respectively.</p