Characterization of multicopper oxidase genes in the green rice leafhopper, Nephotettix cincticeps (Hemiptera: Cicadellidae), with focus on salivary gland-specific genes.

Abstract

Multicopper oxidase (MCO) enzymes are present ubiquitously and act on diverse substrates. Recently, the presence of multiple MCO genes has been described in many insects. Based on sialotranscriptome data, we identified and comprehensively characterized six MCO genes: NcLac1S, 1G, and 2-5 in the green rice leafhopper, Nephotettix cincticeps (Hemiptera: Cicadellidae). NcLac1S and NcLac1G belong to the MCO1 ortholog of other insects. NcLac2 forms a clade with MCO2s involved in the sclerotization and pigmentation of the cuticle. NcLac3 and NcLac4 form a clade with NlMCO3 -5 of the hemipteran Nilaparvata luges. NcLac5 forms a clade with MCORPs (MCO-related proteins) that lack amino acid residues normally highly conserved in copper-coordinated MCOs. NcLac1S and NcLac3 were specifically expressed in the salivary glands; whereas NcLac5 was primarily expressed in the salivary glands. Only NcLac3 protein is considered to have laccase activity in the salivary glands and salivary sheaths ejected by the insect. NcLac1G expression was relatively high in the testis. NcLac2 and NcLac4 were specifically expressed in the integument and in Malpighian tubules, respectively. Knockdown by RNA interference (RNAi) of either NcLac2 and NcLac5 in nymphs caused high mortality. All NcLac2-knockdown nymphs showed depigmentation and soft cuticle, and eventually died, as did other MCO2-knockdown insects. DsNcLac5-injected nymphs (third, fourth, and fifth-instar) showed high mortality, but injection into adults had no effect on survival or number of eggs deposited, suggesting that NcLac5 is not essential for survival after molting (eclosion). NcLac5 could be a promising target gene for control of N. cincticeps.