Anopheles coluzziistearoyl-CoA desaturase is essential for adult female survival and reproduction upon blood feeding

Abstract

Vitellogenesis and oocyte maturation require anautogenous female Anopheles mosquitoes to obtain a bloodmeal from a vertebrate host. The bloodmeal is rich in proteins that are readily broken down into amino acids in the midgut lumen and absorbed by the midgut epithelial cells where they are converted into lipids and then transported to other tissues including ovaries. The stearoyl-CoA desaturase (SCD) plays a pivotal role in this process by converting saturated (SFAs) to unsaturated (UFAs) fatty acids; the latter being essential for maintaining cell membrane fluidity amongst other housekeeping functions. Here, we report the functional and phenotypic characterization of SCD1 in the malaria vector mosquito Anopheles coluzzii. We show that RNA interference (RNAi) silencing of SCD1 and administration of sterculic acid (SA), a small molecule inhibitor of SCD1, significantly impact on the survival and reproduction of female mosquitoes following blood feeding. Microscopic observations reveal that the mosquito thorax is quickly filled with blood, a phenomenon likely caused by the collapse of midgut epithelial cell membranes, and that epithelial cells are depleted of lipid droplets and oocytes fail to mature. Transcriptional profiling shows that genes involved in protein, lipid and carbohydrate metabolism and immunity-related genes are the most affected by SCD1 knock down (KD) in blood-fed mosquitoes. Metabolic profiling reveals that these mosquitoes exhibit increased amounts of saturated fatty acids and TCA cycle intermediates, highlighting the biochemical framework by which the SCD1 KD phenotype manifests as a result of a detrimental metabolic syndrome. Accumulation of SFAs is also the likely cause of the potent immune response observed in the absence of infection, which resembles an auto-inflammatory condition. These data provide insights into mosquito bloodmeal metabolism and lipid homeostasis and could inform efforts to develop novel interventions against mosquito-borne diseases.ClustalW and protein prediction was done using the SMART Package (http://smart.emblheidelberg.de/). Pathway analysis of transcriptomic and metabolomic data was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG). DsRNA preparation and gene silencingGene-specific double-stranded RNA (dsRNA) was synthesized and injected as described [12]. Briefly, SCD1-specific oligonucleotide primers flanked at their 5′ end by T7 RNA polymerase promoter sequence (SCD1-RNAi-F: taatacgactcactataggg-AAATGTGATTGCCTTCGGT; SCD1-RNAi-R: taatacgactcactataggg-GCGAGAAGAAGAAGCCAC) were used in PCR reactions on total RNA extracted from 10 sugar-fed adult female mosquitoes using the TRIzol reagent (Invitrogen). DsRNA was synthesized using the T7 MEGAscript kit (Invitrogen) and the PCR product was purified using the RNeasy kit (QIAGEN). DsRNA for the control LacZ gene was synthesized as reported [13]. Gene silencing was accomplished by injection of 69 nL (3 ug/uL) of dsRNA into the mesothoracic spiracle of each carbon dioxide-anesthetized 0-2-day old female mo...