Bioinformatic analysis and functional study of nkd gene in larvae of Apis mellifera ligustica workers

Abstract:

[Objective] Apis mellifera ligustica is one of the subspecies of Apis mellifera. Ascosphaera apis exclusively infects bee larvae and leads to chalkbrood. In this study, we predicted the conserved motifs and analyzed the phylogenetic relationship of naked cuticle (Nkd) protein in A. mellifera. Further, we employed RNA interference (RNAi) to study the effect of nkd gene on the larval body weight of A. m. ligustica and the immune response to A. apis stress, aiming to enrich the information of A. mellifera nkd gene and reveal the role of nkd in A. m. ligustica larvae. [Methods] The conserved motifs of Nkd proteins from A. mellifera and nine other species were predicted by MEME. MEGA X was used for the phylogenetic analysis of these Nkd proteins. RNAi of the nkd in the guts of the worker larvae of A. m. ligustica was carried out by feeding dsRNA-nkd. The larvae were weighed via an electronic balance. RT-qPCR was employed to determine the relative expression of nkd and immune response genes. [Results] The nkd proteins from A. mellifera, Apis cerana, Papilio xuthus, Bombyx mori, and Papilio machaon contained three conserved motifs (motif 1, motif 2, and motif 3), which indicated that the Nkd proteins from the above-mentioned five insect species were highly conserved. The Nkd proteins in A. mellifera and A. cerana shared the same clade, showing the closest genetic relationship. Compared with the dsRNA-egfp group, the dsRNA-nkd group showed down-regulated expression of nkd in the guts of 5- and 6-day-old larvae (P<0.001), with the interference efficiencies of 49.60% and 56.40%, respectively. Additionally, the body weights of all the larvae in the dsRNA-nkd group were significantly lower than those in the dsRNA-egfp group. The expression of abaecin, apidaecin, birc5, defensin-1, and PGRP-S2 was activated in the guts of 4-day-old larvae. In the guts of 5-day-old larvae, the expression of abaecin, apidaecin, birc5, and defensin-1 was activated, while that of PGRP-S2 was inhibited. In the guts of 6-day-old larvae, the expression of abaecin was activated, while that of apidaecin, birc5, defensin-1, and PGRP-S2 was suppressed. The results suggested that the expression of above-mentioned five genes varied in response to stress and were involved in the host immune response. Moreover, there was a negative regulatory relationship between the expression of nkd and that of abaecin and apidaecin. [Conclusion] A. mellifera Nkd protein contains three conserved motifs (motif 1, motif 2, and motif 3). The Nkd proteins from A. mellifera and A. cerana shared the closest genetic relationship. The interference on nkd in the larval guts of A. m. ligustica workers could be achieved via feeding dsRNA, and nkd affected A. m. ligustica workers in terms of larval body weight and immune response to A. apis stress.

Key words:Apis mellifera;Apis mellifera ligustica;Ascosphaera apis;RNA inference;naked cuticle protein;function;immune response