Nucleoplasmin-Like Protein, Expression Differences During the Caste Polyphenism Processes in Apis mellifera

In honey bees, queen-worker differentiation is regulated by diet and begins after the first 3 days post-hatching. Several molecules and processes appear to be involved in caste polyphenism, including juvenile hormone, royalactin, and DNA methylation. A protease (PS4) was recently detected in worker larvae at 72 h post-hatching and shown to be inhibited by royal jelly, suggesting a potential role in this process. This study investigated the expression and tissue localization of nucleoplasmin during caste polyphenism in Apis mellifera larvae. A protein spot corresponding to a previously described caste-specific proteolytic activity was separated by 2DE and identified by mass spectrometry as a nucleoplasmin-like protein. Based on this result, a polyclonal antibody was raised against a conserved nucleoplasmin peptide and subsequently used for immunoproteomic and immunohistochemical analyses in worker- and queen-destined larvae. At this instar, Western blot analysis using a polyclonal anti-nucleoplasmin-like protein antibody revealed bands between 43-88 kDa and 74-104 kDa in worker- and queen-destined larvae, respectively, which may correspond to different polymeric conformations of the nucleoplasmin-like protein. Differences in the molecular mass of nucleoplasmin-like protein spots between worker and queen larvae suggest caste-specific roles during differentiation. Immunohistochemistry showed a ubiquitous nuclear distribution of the protein in undifferentiated larvae. Worker-fate larvae diverge from this pattern as early as Day 4, whereas queen-fate larvae retain a distribution like that of undifferentiated larvae, possibly representing the baseline developmental condition. Further studies examining temporal changes in nucleoplasmin-like protein expression between the third and fourth larval instars and during post-larval stages are desirable.