Fig 1.
Gene and transcript structure of Ae. albopictus Piwi5 and Piwi7.
A) Schematic representation of the DNA structure of Piwi5 and Piwi7 genes and their corresponding transcripts as obtained from cDNA amplification of single sugar-fed mosquito samples. Exons and introns are shown by blue boxes and black lines, respectively, with corresponding length in nucleotide below each. The positions of the predicted PAZ, MID and PIWI domains are shown by green, blue and magenta ovals, respectively. Exon numbers correspond to lane numbers. B) Amplification of each exon of Piwi5 and Piwi7 on genomic DNA. Exon numbers correspond to lane numbers. C) Northern-blot results of Piwi5 indicate the presence of a transcript of 3 kb.
Fig 2.
Venn diagrams showing the number of positions harbouring synonymous and non-synonymous mutations in tested samples for each Piwi gene.
Table 1.
Polymorphism of Aedes albopictus Piwi genes in mosquitoes from the Foshan strain and wild-caught mosquitoes from La Reunion (Reu) and Mexico (Mex).
We report the number of sequences (n), as well as the number of sites (L), segregating sites (S), polymorphism measured as π and θ, and the Tajima’s D statistic for both synonymous (s) and non-synonymous sites (a) for each gene and population (and for the pooled sample).
Table 2.
Insights into Evolutionary divergence of Piwi genes in Ae. albopictus.
A) McDonald-Kreitman test for each Piwi gene using the orthologous sequences of Ae. aegypti as outgroup. NI = Neutrality Index; Alpha = proportion of base substitutions fixed by natural selection; P estimated using Fisher’s exact test. B) Output of Codeml with significant results regarding sites under positive selection.
Fig 3.
Level of polymorphism (LoP) comparison between slow-evolving genes (SGs), fast-evolving genes (FGs) and Piwi genes by population. Genes on the right side of the panel have LoP values greater than those of SGs, while genes on the left side have smaller LoPs than SGs. The y-axis represents the -log10 p-values of the Kolmogorov-Smirnov test. Faint datapoints are not significant after Bonferroni correction for multiple testing (-log10 0.0024 (0.05/21 genes) = 2.62).
Fig 4.
Computational homology models of the Ae. Albopictus Piwi proteins.
Homology models were generated for the seven Piwi genes as described in the methods section. A) Superposition of cartoon representations of Piwi homology models, with highlight of domain organization: the N-terminal domain is shown in orange, the PAZ domain in green, the MID domain in blue and the PIWI domain in magenta. B) CONSURF [46]overview of the aminoacid sequence conservation mapped on three-dimensional homology models in a putative RNA-bound arrangement based on the structure of human Argonaute bound to a target RNA (PDB ID 4Z4D), colored from teal (very low conservation) to dark magenta (highly conserved).
Fig 5.
Expression profile of Piwi genes.
Heatmap representations of log10 transformed fold-change expression values of each Piwi gene. A) Developmental expression pattern of the Piwi genes normalized on the expression in sugar fed females. B) Expression pattern of Piwi genes following viral infection normalized with respect to sugar-fed samples. Expression was verified in ovaries and carcasses separately, during the early and late stages of infections, that is 4 dpi for both viruses and 14 or 21 dpi for CHIKV and DENV, respectively. Each day post-infection was analysed with respect to sugar and blood-fed controls of the same day. * indicates significant difference (P<0.05) between infected samples and the corresponding blood-fed control.