3.3 A gene expression profile of male die-off inAntechinus
We profiled gene expression during antechinus semelparity by surveying
multiple tissues from an A. flavipes population in Samford Valley
in Queensland, Australia during the 2018 breeding season (File
S1 ). We observed reproductive behaviour and peak oestrus on September
15, 2018 (see Supplemental Methods for details). Adult male A.
flavipes disappear from this site after the first week of October.
Antechinus male die-off is characterised by a dramatic increase in free
cortisol (Figure 3a ), mediated by a surge in testosterone which
decreases the production of corticosteroid-binding globulin (Boonstra,
2005; Naylor et al., 2008). The potent effects of cortisol mean that the
associated gene expression and its consequences (McDowell et al., 2018;
Phuc Le et al., 2005) become a suitable backdrop against which
comparisons can be made. While corticosteroids regulate a large number
of genes via the glucocorticoid receptor (GR) (McDowell et al., 2018;
Phuc Le et al., 2005), complicating gene expression analyses, chronic
(i.e., breeding male antechinus) exposure to free corticosteroids has
distinct outcomes (Manoli et al., 2007). The steroid hormone mediates a
stress response, alters metabolism and mobilises glucose as energy from
a series of stores, initially enhancing short-term survival. Because of
a limited number of samples in our current data set, we identified genes
differentially expressed by moribund antechinus males; individuals
sampled near the conclusion of breeding, in early October, when all
males die (right panel in Figure 3b ).
There were more differentially expressed genes (limma-modified Student’st -test P ≤0.01) in the liver and skeletal muscle than the
kidney and cerebrum (Figure 3c and File S3 ), and most
of these were downregulated. Genes upregulated in moribund male livers
showed enrichment of biological process gene sets attributed to the
innate immune response (bootstrap P ≤0.0001) (Table S9 ).
However, several cytokine genes (PDGFRA , PDGFRB ,PRNP , CD248 , CD79A , CXCR5 , and CXCR6 )
were downregulated in the moribund liver (File S3 ).
Characteristic liver pathology of the moribund male antechinus includes
infections by bacteria such as Listeria monocytogenes (Barker,
Beveridge, Bradley, & Lee, 1978; A. Bradley, McDonald, & Lee, 1980),
viruses (e.g., dasyurid herpesvirus 1 (Amery-Gale et al., 2014)), and
parasitic infestation (Poskitt et al., 1984). Cortisol-mediated
immunosuppression in the preceding two weeks is likely to lead to poor
late-stage resistance to invading pathogens. We identified eight genes
that were downregulated and two genes that were upregulated by both
liver and skeletal muscle of moribund males (Figure 3d ). This
included decreased expression of the glycophagy receptor STBD1 (Johansen
& Lamark, 2020) and increased expression of ASNS – a gene
induced by amino acid starvation stress (Balasubramanian, Butterworth,
& Kilberg, 2013). Downregulation of SLC2A4 (encodes the glucose
transporter GLUT4 (Amoasii et al., 2019)) and NUR4A2 (NUR11; a
regulator of GLUT4 expression (Amoasii et al., 2019)) indicates
reduced glucose uptake by the skeletal muscle of moribund males. KEGG
pathway and biological process enrichment analysis revealed metabolic
dysregulation of skeletal muscle in moribund males, with marked
downregulation of genes associated with glycolysis, gluconeogenesis, and
insulin signalling – indicating insulin resistance (bootstrapP ≤0.0001) (Figure 3e , Tables S9 and S10 ). These
genes included PRKAG3 (-4.7-fold; limma-modified Student’st -test, P =3.7 × 10-4), which encodes a
subunit of a key energy-sensing molecule, AMP-activated protein kinase,
and its regulator LKB1 (STK11 ; -2.0-fold, P =2.4 ×
10-3) (File S3 ). AMPK responds to energetic
stress to regulate multiple metabolic pathways. Reduced expression of
LKB1 was limited to the skeletal muscle of moribund A. flavipesmales (Figure 3f ). LKB1 has a tissue-specific role in energy
metabolism. Complete or partial loss of Stk11 expression in mouse
skeletal muscle prevents AMPK activation and causes metabolic defects
such as acceleration of ageing-induced myopathy (Bujak et al., 2015; Koh
et al., 2006; Sakamoto et al., 2005; Thomson et al., 2007). Our work is
the first gene expression profile of a semelparous mammal. In agreement
with observational data from A. flavipes and other antechinuses
(A. Bradley et al., 1980; Naylor et al., 2008; Woollard, 1971), our
results indicate near-complete depletion of fat stores and skeletal
muscle atrophy – leading to a catastrophic energy crisis and an
inability to maintain cognitive and physical function in moribund males.
Further experimental work on the endocrine system of semelparous mammal
species is needed to characterise the role of testosterone and the
dysregulation of the hypothalamic-pituitary-adrenal axis and negative
feedback by cortisol. We were not able to capture gene expression
changes in brain regions other than the whole cerebrum of A.
flavipes during the breeding season, limiting our insights into the
effects on major organs. Nevertheless, the A. flavipes reference
genome permits future studies on gene expression and regulation (e.g.,
glucocorticoid receptor ChIP-seq).