Differential Hepatic Expression of miRNA in Response to Aflatoxin B1 Challenge in Domestic and Wild Turkeys

Abstract

Aflatoxin B1 (AFB₁) is a major foodborne mycotoxin that poses a significant economic risk to poultry due to a greater degree of susceptibility compared to other agricultural species. Domesticated turkeys (Meleagris gallopavo) are especially sensitive to AFB₁; however, wild turkeys (M. g. silvestris) are more resistant. A lack of functional isoforms of hepatic glutathione S-transferases (GSTs), an enzyme that plays a role in the detoxification of aflatoxin, is suspected as the reason for the increased sensitivity. Previous studies comparing the gene expression of domesticated and wild turkeys exposed to AFB₁ identified hepatic genes responding differentially to AFB₁, but could not fully explain the difference in response. The current study examined differences in the expression of microRNAs (miRNAs) in the livers of wild and domesticated turkeys fed dietary AFB₁ (320 μg/kg in feed). Short-read RNA sequencing and expression analysis examined both domesticated and wild turkeys exposed to AFB₁ compared to controls. A total of 25 miRNAs was identified as being significantly differentially expressed (DEM) in pairwise comparisons. The majority of these have mammalian orthologs with known dysregulation in liver disease. The largest number of DEMs occurred between controls, suggesting an underlying difference in liver potential. Sequences of the DEMs were used to identify potential miRNA binding sites in target genes, resulting in an average of 4302 predicted target sites per DEM. These DEMs and gene targets provide hypotheses for future investigations into the role of miRNAs in AFB₁ resistance.

Keywords: RNA-seq; aflatoxin B1; domesticated turkey; liver; microRNA; wild turkey.

Figure 1

(A). Distribution of sequencing reads (Millions of fragments) in sequencing libraries of treatment groups (Control and AFB₁). (B). Principal component analysis (PCA) plots of normalized read counts. Sample-to-sample distances (within- and between-treatments) are illustrated for each treatment sample on the first two principal components and plotted according to treatment. (C). Distribution of sample variance by treatment factor: Genotype (Line, Eastern Wild (EW) and Nicholas (NT) turkeys), AFB₁ treatment, interaction, and residual.

Figure 2

UpSet plot of expressed miRNAs in turkey liver. For inclusion, miRNAs must first have at least three assigned reads in at least two libraries and, secondly, a treatment group with an average number of reads > 2.0. The horizontal bars on the left (Set size) indicate the number of miRNAs expressed in each treatment. Individual points in the matrix represent miRNAs expressed in each treatment, and the lines between points represent shared expression. The vertical bars above indicate the number of miRNAs specific to or common to different treatments.