Long-chain acyl-CoA synthetase 2 knockdown leads to decreased fatty acid oxidation in fat body and reduced reproductive capacity in the insect Rhodnius prolixus.
Long-chain acyl-CoA esters are important intermediates in lipid metabolism and are synthesized from fatty acids by long-chain acyl-CoA synthetases (ACSL). The hematophagous insect Rhodnius prolixus, a vector of Chagas' disease, produces glycerolipids in the midgut after a blood meal, which are stored as triacylglycerol in the fat body and eggs. We identified twenty acyl-CoA synthetase genes in R. prolixus, two encoding ACSL isoforms (RhoprAcsl1 and RhoprAcsl2). RhoprAcsl1 transcripts increased in posterior midgut on the second day after feeding, and RhoprAcsl2 was highly transcribed on the tenth day. Both enzymes were expressed in Escherichia coli. Recombinant RhoprACSL1 and RhoprACSL2 had broad pH optima (7.5-9.5 and 6.5-9.5, respectively), were inhibited by triacsin C, and were rosiglitazone-insensitive. Both showed similar apparent Km for palmitic and oleic acid (2-6 μM), but different Km for arachidonic acid (0.5 and 6 μM for RhoprACSL1-Flag and RhoprACSL2-Flag, respectively). The knockdown of RhoprAcsl1 did not result in noticeable phenotypes. However, RhoprACSL2 deficient insects exhibited a 2.5-fold increase in triacylglycerol content in the fat body, and 90% decrease in fatty acid β-oxidation. RhoprAcsl2 knockdown also resulted in 20% increase in lifespan, delayed digestion, 30% reduced oviposition, and 50% reduction in egg hatching. Laid eggs and hatched nymphs showed remarkable alterations in morphology. In summary, R. prolixus ACSL isoforms have distinct roles on lipid metabolism. Although RhoprACSL1 functions remain unclear, we propose that RhoprACSL2 is the main contributor for the formation of the intracellular acyl-CoA pool channeled for β-oxidation in the fat body, and is also required for normal reproduction.
Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; Department of Nutrition, University of North Carolina, Chapel Hill, NC 27599, USA.
Michele
M
Alves-Bezerra
Department of Nutrition, University of North Carolina, Chapel Hill, NC 27599, USA; Department of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA.
Eric L
EL
Klett
Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
Iron F
IF
De Paula
Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
Isabela B
IB
Ramos
Department of Nutrition, University of North Carolina, Chapel Hill, NC 27599, USA.
Rosalind A
RA
Coleman
Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. Electronic address: katia@bioqmed.ufrj.br.
Katia C
KC
Gondim
D000595:Amino Acid Sequence; D000818:Animals; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005216:Fat Body / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D019476:Insect Proteins; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009866:Oogenesis / Q000235:genetics*; D010084:Oxidation-Reduction; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012098:Reproduction / Q000235:genetics; D012239:Rhodnius / Q000145:classification / Q000235:genetics*; D016415:Sequence Alignment; D014158:Transcription, Genetic; D014226:Triazenes; D014280:Triglycerides / Q000096:biosynthesis*; D015053:Zygote / Q000378:metabolism; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
D005227:Fatty Acids; D019476:Insect Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D014226:Triazenes; D014280:Triglycerides; C034613:triacsin C; D003066:Coenzyme A Ligases; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
Acyl-CoA metabolism; Insect reproduction; Lipid synthesis; Oogenesis; β-Oxidation