Abstract
Ants are highly diverse social insects living in colonies consisted of up to millions of individuals with reproductive division of labors. Due to the interests in disclosing the genetic and epigenetic regulation mechanisms underlying the distinct developmental trajectories between castes and division of labor in colonies, many ant species have recently been established as laboratory models for evolutionary development and social behavior studies. These functional studies often request a precise quantification of the relative gene expression level, which relies on a stably expressed reference genes for normalization. A core set of reliable reference genes for this purpose however has not been established yet in ants. In the present study, we tested the expression patterns and amplification efficiencies of 12 abundantly expressed candidate genes in Monomorium pharaonis, one of the few ant species that are suitable for laboratory rearing and experimentation. We quantified the expression levels of these genes by RT-qPCR in seven different conditions: embryo development, sexual development, worker development, adult phenotypes, tissues, and two abiotic manipulative treatments in pharaoh ant. Finally, five genes, elongation factor-1 alpha (EF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), TATA-box-binding protein (TATA), tubulin gamma-2 chain-like (TBLg2), heat shock protein 67B2-like (HSP67) were found to be the most stable reference genes across seven conditions. We also identified the most stable reference genes applicable for each distinct condition and the optimal number of reference genes entailed were evaluated. Our study validates reliable reference genes for RT-qPCR analysis which lays the foundation for future studies in pharaoh ant.
Highlights
Social insects live in complex societies with sophisticated social behavior and division of labor
In our RNA-seq data, none of the genes was annotated as 18S ribosomal RNA (18S) and SDHA, two of the top 10 candidate genes in insects; 205 genes were annotated as the homologs of the other eight reference genes (Supplementary Table 1)
Addition to 18S, annotated in NCBI, and NADH, validated in ants, we obtained a list of 12 candidate reference genes for the subsequent experiments (Table 1)
Summary
Social insects live in complex societies with sophisticated social behavior and division of labor. The differentiated castes vary in morphology, physiology, and behavior even though they have the same genome (Abouheif and Wray, 2002; Li et al, 2014). With the morphologically and behaviorally distinct individuals, ant colony organizes into a complicated society with intricate division of labor among different castes (Hölldobler and Wilson, 1990). Many studies suggested that the caste differentiation is regulated by differentially expressed genes among castes (Evans and Wheeler, 2002; Pereboom et al, 2005; Khila and Abouheif, 2010; Qiu et al, 2018). Interrogating the molecular mechanism underlying caste differentiation through the gene expression analysis has been the major focus in recent studies in social insects (Gospocic et al, 2017; Chandra et al, 2018)
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