Journal: bioRxiv

Article Title: High-Resolution Proteomics Unveils Salivary Gland Disruption and Saliva-Hemolymph Protein Exchange in Plasmodium -Infected Mosquitoes

doi: 10.1101/2025.02.28.640873

Figure Lengend Snippet: (A) Relative mRNA expression of selected classical saliva genes by qRT-PCR analyses in uSGs and iSGs 20 days post-infection. Antigen 5-related protein 1 (A5R1), apyrase (Apy), Anopheles antiplatelet protein (AAPP), 5’nucleotidase ecto (NT5E), D7-related protein 3 (D7R3). Relative expression levels were calculated using the delta-delta Ct (ΔΔCt) method, with ribosomal protein S7 used as the reference gene for normalization. (B) Selected proteins showing differential expression between uSGs and iSGs. Transferrin 1 (Transf), lipophorin (LP), clip domain serine protease related protein A 14 (CLIPA14) and prophenoloxidase 6 (PPO6). Bars: Mean fold change in gene expression relative to uninfected controls ± SD. Dots: Individual biological replicates. Differences in relative expression between the conditions were determined using an unpaired t-test (Mann-Whitney). Significance levels are indicated as: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. (C). RNA in situ hybridization in uSGs (C) and (D) iSGs. Nuclei in blue, PPO6 in yellow, Apyrase in green and Lipophorin in red. (LL) lateral lobe and (CL) central lobe. Arrow in (C) and (D) indicates fat body cells associated with SGs. (E) Close-up of an iSG. Nuclei in blue, PPO6 in yellow, Apyrase in green and Lipophorin in red. Dashed insert identifies a PPO6-positive hemocyte in the surface of an iSGs. (F) Close-up of a PPO6-positive hemocyte. (G) Lateral view of an iSG with a hemocyte expressing PPO6 RNA. Arrow indicates hemocyte in the surface of the lobe.

Article Snippet: Tissues were blocked with Protein Blocker (X0909, Dako) for 30 min prior to a 60 min incubation with the following primary antibodies: anti-TEP15 rabbit polyclonal serum (1:200, Pacific Immunology), anti-Transferrin 1 rabbit polyclonal serum (1:500, Pacific Immunology), anti-PPO6 rabbit polyclonal serum (1:500) — kindly provided by Dr. Ryan Smith from Iowa State University —, anti-AAPP rabbit polyclonal serum (1:200, Pacific Immunology), anti-Lipophorin (1:400, Pacific Immunology), anti-CLIPA14 (1:400, Boster Bio), and anti-circumsporozoite (CS) protein mouse monoclonal antibody (3D11, 1:1000, Pacific Immunology). .

Techniques: Expressing, Quantitative RT-PCR, Infection, Gene Expression, MANN-WHITNEY, RNA In Situ Hybridization