•An aphid specific cysteine-rich protein is expressed highly in salivary glands.•This protein is a monomer globular molecule with a high extent of beta strand.•This protein is able to bind zinc ions at two binding sites.•Aphids require more of this protein when feeding on plants.Aphids secrete saliva into the phloem during their infestation of plants. Previous studies have identified numerous saliva proteins, but little is known about the characteristics (physical and chemical) and functions of these proteins in aphid–plant interactions. This study characterized an unknown protein (ACYPI39568) that was predicted to be enriched in the salivary glands of pea aphid. This protein belongs to an aphid-specific, cysteine-rich protein family that contains 14 conserved cysteines. ACYPI39568 is a monomeric globular protein with a high beta strand extent. The binding stoichiometric ratios for Zn2 + and ACYPI39568 were approximately 3:1 and 1:1 at two binding sites. ACYPI39568 was predominantly expressed in the first instar stage and in the salivary glands. Aphids required more ACYPI39568 when feeding on plants than when feeding on an artificial diet. However, the interference of ACYPI39568 expression did not affect the survival rate of aphids on plants.

•The ABC transporters of three mosquitoes were classified with phylogenetic analysis.•ABCG and ABCC are the largest subfamilies in mosquitoes.•Subfamilies B, D, E, and F are conserved within the three mosquito species.•Expression profiles of Culex ABC genes along developmental stages were analyzed.The ATP-binding cassette (ABC) transporter family functions in the ATP-dependent transportation of various substrates across biological membranes. ABC proteins participate in various biological processes and insecticide resistance in insects, and are divided into eight subfamilies (A–H). Mosquitoes are important vectors of human diseases, but the mechanism by which the ABC transporter family evolves in mosquitoes is unknown. In this study, we classified and compared the ABC transporter families of three mosquitoes, namely, Anopheles gambiae, Aedes aegypti, and Culex pipiens quinquefasciatus. The three mosquitoes have 55, 69, and 70 ABC genes, respectively. The C. p. quinquefasciatus had approximately 40% and 65% expansion in the ABCG subfamily, mainly in ABCG1/G4, compared with the two other mosquito species. The ABCB, ABCD, ABCE, and ABCF subfamilies were conserved in the three mosquito species. The C. p. quinquefasciatus transcriptomes during development showed that the ABCG and ABCC genes were mainly highly expressed at the egg and pupal stages. The pigment-transport relative brown, white, and scarlet, as well as the ABCF subfamily, were highly expressed at the egg stage. The highly expressed genes in larvae included three ABCA3 genes. The majority of the highly expressed genes in adults were ABCG1/4 genes. These results provided insights into the evolution of the ABC transporter family in mosquitoes.Download full-size image

•ACYPI006346 is an aphid specific and small secretory protein.•ACYPI006346 is enriched in salivary glands and mainly at two cell types.•The dsRNA interference of ACYPI006346 expression lasts for 24 h.•The transcript levels of ACYPI006346 are different in three pea aphid colonies.Salivary proteins play key roles in the co-evolution of aphids and their host plants. This study characterizes the aphid-specific and small secretory protein ACYPI006346 in the pea aphid Acyrthosiphon pisum. The ACYPI006346 transcript is enriched in the salivary glands, particularly in cell types 5 and 7 of the principal salivary glands. Transcript knockdown by dsRNA injection, which lasts for 24 h does not influence the survival of aphids on plants. The transcript levels of ACYPI006346 were different in three pea aphid colonies adapting to Vicia faba, Vicia villosa, or Medicago truncatula, respectively.Download full-size image

•Wild-type and two mutant esterases from two insects are transferred to fruit flies.•G/A151D or W271L mutation disrupts enzyme's original activities.•Mutant esterases bring at most low organophosphate resistance in flies.•Overexpression of any type of esterases causes low insecticide resistance.Carboxylesterases are mainly involved in the mediation of metabolic resistance of many insects to organophosphate (OP) insecticides. Carboxylesterases underwent two divergent evolutionary events: (1) quantitative mechanism characterized by the overproduction of carboxylesterase protein; and (2) qualitative mechanism caused by changes in enzymatic properties because of mutation from glycine/alanine to aspartate at the 151 site (G/A151D) or from tryptophan to leucine at the 271 site (W271L), following the numbering of Drosophila melanogaster AChE. Qualitative mechanism has been observed in few species. However, whether this carboxylesterase mutation mechanism is prevalent in insects remains unclear. In this study, wild-type, G/A151D and W271L mutant carboxylesterases from Culex pipiens and Aphis gossypii were subjected to germline transformation and then transferred to D. melanogaster. These germlines were ubiquitously expressed as induced by tub-Gal4. In carboxylesterase activity assay, the introduced mutant carboxylesterase did not enhance the overall carboxylesterase activity of flies. This result indicated that G/A151D or W271L mutation disrupted the original activities of the enzyme. Less than 1.5-fold OP resistance was only observed in flies expressing A. gossypii mutant carboxylesterases compared with those expressing A. gossypii wild-type carboxylesterase. However, transgenic flies universally showed low resistance to OP insecticides compared with non-transgenic flies. The flies expressing A. gossypii W271L mutant esterase exhibited 1.5-fold resistance to deltamethrin, a pyrethroid insecticide compared with non-transgenic flies. The present transgenic Drosophila system potentially showed that a quantitative increase in carboxylesterases induced broader resistance of insects to insecticides than a qualitative change.Download high-res image (60KB)Download full-size image