Immune profiles of male giant panda (Ailuropoda melanoleuca) during the breeding season

Abstract

Background: The giant panda (Ailuropoda melanoleuca) is a threatened endemic Chinese species and a flagship species of national and global conservation concern. Life history theory proposes that reproduction and immunity can be mutually constraining and interrelated. Knowledge of immunity changes of male giant pandas during the breeding season is limited.

Results: Here, we researched peripheral blood gene expression profiles associated with immunity. Thirteen captive giant pandas, ranging from 9 to 11 years old, were divided into two groups based on their reproductive status. We identified 318 up-regulated DEGs and 43 down-regulated DEGs, which were enriched in 87 GO terms and 6 KEGG pathways. Additionally, we obtained 45 immune-related genes with altered expression, mostly up-regulated, and identified four hub genes HSPA4, SUGT1, SOD1, and IL1B in PPI analysis. These 45 genes were related to pattern recognition receptors, autophagy, peroxisome, proteasome, natural killer cell, antigen processing and presentation. SUGT1 and IL1B were related to pattern recognition receptors. HSP90AA1 was the most up-regulated gene and is a member of heat shock protein 90 family. HSP90 contributes to the translocation of extracellular antigen. KLRD1 encodes CD94, whose complex is an inhibitor of the cytotoxic activity of NK cells, was down-regulated. IGIP, which has the capability of inducing IgA production by B cells, was down-regulated, suggesting low concentration of IgA in male giant pandas. Our results suggest that most immune-related genes were up-regulated and more related to innate immune than adaptive immune.

Conclusions: Our results indicated that breeding male giant pandas presented an immunoenhancement in innate immunity, enhanced antigen presentation and processing in cellular immunity compared to non-breeding males. The humoral immunity of male giant pandas may show a tendency to decrease during the breeding season. This study will provide a foundation for further studies of immunity and reproduction in male giant pandas.

Keywords: Breeding season; Immune change; Male giant panda; RNA-seq.

Fig. 1

PCA analysis of 13 samples

Fig. 2

Partial GO enrichment of up-regulated DEGs

Fig. 3

GO enrichment of down-regulated DEGs

Fig. 4

KEGG enrichment of up-regulated and down-regulated DEGs

Fig. 5

Summary of the main categories and subcategories of KEGG pathways analyzed by DIA. On the right are the bar denoting the overall impact (in blue) and the shade denoting the effect on the pathway (from green (inhibited)—to red (activated)). Darker the color larger the activation (if red) or inhibition (if green) of the pathway. “B” represents the “breeding season”. “non-B” represents the “non-breeding season”

Fig. 6

The 20 most impacted pathways analyzed by DIA. On the right are the bar denoting the overall impact (in blue) and the shade denoting the effect on the pathway (from green (inhibited)—to red (activated)). Darker the color larger the activation (if red) or inhibition (if green) of the pathway. “B” represents the “breeding season”. “non-B” represents the “non-breeding season”

Fig. 7

Chord diagram of categories of immune-associated genes. Genes were divided into twelve branches according to KEGG pathways: A –Antigen processing and presentation; B –Autophagy; C –C-type lectin receptor signaling pathway; D –Cytokine and Chemokine; E –IL-17 signaling pathway; F –Natural killer cell mediated cytotoxicity; G –NOD-like receptor signaling pathway; H –Peroxisome; I –Proteasome; J –T cell receptor signaling pathway; K –Th17 cell differentiation; L –TNF signaling pathway

Fig. 8

Heat map plot of 45 immune-related genes. The expression values of 13 pandas are presented after being centered and scaled in the row direction. Each column represents a specimen and each row represents a gene. Red color indicates genes which were up-regulated and green color indicates genes which were down-regulated

Fig. 9

Protein-protein interaction network of DEGs. The nodes represent proteins and edges represent pair-wise interactions. The size of the nodes is score of the protein calculated by cytoHubba. The red nodes represent up-regulated proteins. The blue nodes represent down-regulated proteins

Fig. 10

The expression level of genes verified by qRT-PCR. “*” represents the P-value< 0.05, “**” represents the P-value< 0.01, “***” represents the P-value< 0.001. Data were shown as mean ± SD. The left axis represents gene expression levels verified by qRT-PCR. The right axis represents the expression levels in TPM units of RNA-seq