A novel eukaryotic Na+ methionine selective symporter is essential for mosquito development

Abstract

AeNAT5 (NCBI, ABZ81822), an orphan member of the insect-specific Nutrient Amino acid Transporter subfamily of SoLute Carrier family 6 (NAT-SLC6) and the first representative of a novel eukaryotic methionine-selective transport system (M), was cloned from cDNA of the vector mosquito, Aedes aegypti. It has orphan orthologs throughout several mosquito genomes, but not in Drosophila or outside Diptera. It shows the highest apparent affinity to L-Met (K0.5 = 0.021 mM) and its metabolites Homocysteine and Cysteine (K0.5 = 0.89 and 2.16 mM), but weakly interact with other substrates. It has a Na+ - coupled mechanism (K0.5 Na+ ~ 46 mM) with 1AA:1Na+ stoichiometry that maintains ~ 60% activity in Cl− - free media. In situ hybridization showed accumulation of AeNAT5 transcript in the absorptive and secretory epithelia, as well as in specific peripheral neurons and the central ganglia of mosquito larvae. The labeling pattern is distinct from that of the previously characterized AeNAT1. RNAi of AeNAT5 increases larval mortality during ecdysis and dramatically suppresses adult emergence. Our results showed that in addition to previously characterized broad spectra and aromatic amino acid selective transport systems, the mosquito NAT-SLC6 subfamily evolved a unique mechanism for selective absorption of sulfur-containing substrates. We demonstrated specific patterns of alimentary and neuronal transcription of AeNAT5 in mosquito larvae that is collateral with the indispensable function of this transporter in mosquito development.

Knockdown of a nutrient amino acid transporter gene LdNAT1 reduces free neutral amino acid contents and impairs Leptinotarsa decemlineata pupation

“…In insects, several SLC6 NATs have been cloned from Lepidopteran species Manduca sexta 5 6 7 8 9 10 , and Dipteran species Aedes aegypti 11 , Anopheles gambiae 12 and Drosophila melanogaster 13 . When expressed in Xenopus oocytes, these NATs can absorb a subset of neutral amino acids 14 . However, in vivo data have not yet been reported.…”

Methionine as a safe and effective novel biorational mosquito larvicide

“…The activity of the responsible enzyme, methionine synthetase, was upregulated in the presence of filarial worms, Brugia pahangi (Buckley & Edeson, 1956 and Buckley, 1960), indicating that adults are able to, at least partially, compensate for diets lower in methionine or increased need for methionine. Furthermore, Meleshkevitch et al . demonstrated the indispensable role of an alimentary and neuronal methionine selective transporter required for mosquito development.…”

RNA Interference for Mosquito and Mosquito-Borne Disease Control

“…For instance, suppression of a Na + methionine symporter ( AeNat5 ) in Ae. aegypti embryos resulted in larval mortality but no impact on embryos was measured [118]. In the context of delivery, the chorion may limit direct delivery of RNAi triggers; that said, there is one example of non-invasive RNAi trigger delivery to eggs via soaking [118].…”

“…aegypti embryos resulted in larval mortality but no impact on embryos was measured [118]. In the context of delivery, the chorion may limit direct delivery of RNAi triggers; that said, there is one example of non-invasive RNAi trigger delivery to eggs via soaking [118]. In this case, eggs were incubated in dsRNA until the time of hatching resulting in 56%–90% reduction in target gene expression in a dose-dependent manner [118].…”

“…In the context of delivery, the chorion may limit direct delivery of RNAi triggers; that said, there is one example of non-invasive RNAi trigger delivery to eggs via soaking [118]. In this case, eggs were incubated in dsRNA until the time of hatching resulting in 56%–90% reduction in target gene expression in a dose-dependent manner [118]. Knockdown in the embryo also has been demonstrated indirectly through plasmid expression vectors [119,120].…”