Mitochondrial Proteins Differential Expression during Honeybee (Apis mellifera L.) Queen and Worker Larvae Caste Determination

Abstract

Despite their similar genetic makeup, honeybee (A. mellifera) queens and workers show alternative morphologies driven by nutritional difference during the larval stage. Although much research have been done to investigate the causes of honeybee caste polymorphism, information at subcellular protein levels is limited. We analyzed queen- and worker-destined larvae mitochondrial proteome at three early developmental stages using combinations of differential centrifugation, two-dimensional electrophoresis, mass spectrometry, bioinformatics, and quantitative real time PCR. In total, 67, 69, and 97 protein spots were reproducibly identified as mitochondrial proteins at 72, 96, and 120 h, respectively. There were significant qualitative and quantitative protein expression differences between the two castes at three developmental stages. In general, the queen-destined larvae up-regulated large proportions of proteins at all of the developmental stages and, in particular, 95% at 72 h. An overwhelming majority of the queen larvae up-regulated proteins were physiometabolic-enriched proteins (metabolism of carbohydrate and energy, amino acid, and fatty acid) and involved in protein folding, and this was further verified by functional enrichment and biological interaction network analyses as a direct link with metabolic rates and cellular responses to hormones. Although wide-ranging mitochondrial proteomes participate to shape the metabolic, physiologic, and anatomic differences between the two castes at 72 h, physiometabolic-enriched proteins were found as the major modulators of the profound marking of this caste differentiation. Owing to nutritional difference, prospective queen larvae showed enhanced growth, and this was manifested through the overexpression of metabolic enzymes. Differently from similar studies targeting the causes of honeybee caste polymorphism, this subcellular level study provides an in-depth insight into mitochondrial proteins-mediated caste polymorphism and greatly improves protein coverage involved during honeybee caste determination. Hence, it is a major step forward in the analysis of the fundamental causes of honeybee caste pathway decision and greatly contributes to the knowledge of honeybee biology. In particular, the consistency between the 22 proteins and mRNA expressions provides us important target genes for the reverse genetic analysis of caste pathway modulation through RNA interference.

Sphingolipids, Transcription Factors, and Conserved Toolkit Genes: Developmental Plasticity in the Ant Cardiocondyla obscurior

“…This is in contrast to studies done in A. mellifera that suggest an increase in energy generation in queen-destined larvae ( Corona et al 1999 ; Li et al 2010 ; Begna et al 2011 ; Cameron et al 2013 ). However, in honeybees, growth rates of queen-destined larvae predominantly increase late in development and queens develop faster than workers ( Page and Peng 2001 ).…”

“…Components of this secretion, including Royalactin, Major Royal Jelly proteins, and a fatty acid Histone Deacetylase inhibitor, likely activate epidermal growth factor receptor (EGFR) signaling in the larval fat body ( Kamakura 2011 ), which in turn activates downstream pathways that directly or indirectly affect developmental trajectories (e.g., insulin-like signaling, PI3K/TOR/S6K, Ras/Raf/MAPK; Patel et al 2007 ; Kamakura 2011 ; Mutti et al 2011 ; Wolschin et al 2011 ; Badisco et al 2013 ). Experimental gene knock-downs, as well as comparative transcriptome and methylome studies, performed mostly in honeybees, have revealed extensive caste-dependent changes in gene expression and epigenetic regulation ( Kucharski et al 2008 ; Elango et al 2009 ; Bonasio et al 2012 ; Shi et al 2012 ; Simola, Ye, et al 2013 ) for a wide range of genes, for example, hexamerins ( Hoffman and Goodisman 2007 ; Hunt et al 2007 ; Cameron et al 2013 ), cytochrome P450s, spliceosomal genes ( Cameron et al 2013 ), the hypoxia pathway ( Azevedo et al 2011 ), and several other metabolic and developmental genes ( Evans and Wheeler 2001 ; Barchuk et al 2007 ; Hoffman and Goodisman 2007 ; Li et al 2010 ; Begna et al 2011 ).…”

Genome-Wide Differential DNA Methylation in Reproductive, Morphological, and Visual System Differences Between Queen Bee and Worker Bee (Apis mellifera)

“…The sampling times were at 3, 4, and 5 days post hatching from eggs (5 queens and 10 workers/day). The detailed sampling method is referred to the reports of Li et al (2010) and Begna et al (2011 , 2012) . DNA was Isolated using the Universal Genomic DNA Extraction Kit (TaKaRa, DV811A).…”

Probiotic (Enterococcus faecium) induced responses of the hepatic proteome improves metabolic efficiency of broiler chickens (Gallus gallus)

“…The identification of protein spots was carried out as described by Begna et al with some modifications [ 22 ]. Interesting protein spots from the preparative gels were in-gel digested and identified by LC-Chip ESI-QTOF-MS (Q-TOF 6520, Agilent, Santa Clara, CA).…”