Interacting effects of sex and age on immune responses in a polygynous bat with male-biased mortality

1. Problem Statement

Bats are renowned for their extraordinary longevity and ability to coexist with highly virulent pathogens (e.g., Ebola, MERS-CoV) without severe pathology. While previous research has focused on interspecific comparisons to identify genomic adaptations (e.g., dampened inflammation, constitutive interferon expression), there is a significant gap in understanding intraspecific variation in immune responses. Specifically, it is unclear how individual factors such as sex, age, and social status influence immune function in wild populations. This is critical because bats exhibit diverse life histories, including extreme polygyny and male-biased mortality, which may drive trade-offs between reproduction, somatic maintenance, and immunity. The study aims to determine if these life-history traits correlate with distinct immune transcriptomic profiles and whether they contribute to immunosenescence (age-related immune decline).

2. Methodology

The study utilized a combination of field sampling, ex vivo immune stimulation, and high-throughput sequencing on the greater spear-nosed bat (Phyllostomus hastatus), a species with a harem-polygynous mating system.

• Sampling: Blood samples were collected from wild bats in Trinidad (N=166 total).
  • Untreated Group (N=70): Blood immediately preserved to establish baseline transcriptomes.
  • LPS-Treated Group (N=96): Blood incubated ex vivo with lipopolysaccharide (LPS) for 3 hours to simulate bacterial infection and provoke an innate immune response.
• Age Estimation: Chronological age was estimated for individuals without mark-recapture records using a DNA methylation clock derived from wing skin biopsies.
• Social Status: Males were categorized as either "harem" (dominant, defending females) or "bachelor" (subordinate).
• Sequencing & Analysis:
  • RNA-seq: Whole-blood transcriptomes were sequenced (Illumina NextSeq 1000).
  • miRNA-seq: Small RNA sequencing was performed on a subset (N=40) to investigate post-transcriptional regulation.
  • Comparative Analysis: Bat LPS responses were compared against public datasets from rhesus macaques, pigs, and yellow baboons.
  • Statistical Modeling: Differential expression analysis (DESeq2), Principal Component Analysis (PCA), and Weighted Gene Co-expression Network Analysis (WGCNA) were employed. Models controlled for sequencing batch, age, sex, and social status. Neutrophil-to-lymphocyte ratio (NLR) was used as a covariate to account for leukocyte composition differences.

3. Key Contributions

• Intraspecific Immune Variation: Demonstrates that individual variation in immune response (driven by sex and age) is substantial and often overlooked in comparative bat immunology.
• Male-Biased Immunosenescence: Provides transcriptomic evidence that males in this polygynous species exhibit accelerated immune aging compared to females, linking this to their shorter lifespans.
• Decoupling Social Status from Immunity: Challenges the prevailing view (derived largely from primates) that social adversity (subordinate status) universally drives pro-inflammatory states, finding no major immune differences between harem and bachelor males in this species.
• Leukocyte Composition: Highlights that observed sex and age differences in gene expression are heavily confounded by shifts in blood cell composition (specifically neutrophil-to-lymphocyte ratios).

4. Key Results

A. Validation of LPS Response

• LPS treatment induced a robust pro-inflammatory response, upregulating 7,394 genes.
• Upregulated genes were enriched for innate immune pathways (TNF, NF-κB, IL-17) and cytokine production.
• Bat responses were largely conserved with other mammals (macaques, pigs, baboons), with 67% of upregulated genes in bats also upregulated in at least one other species.

B. Sex and Age Differences

• Magnitude of Response: Males mounted a stronger inflammatory response to LPS than females. This was evidenced by:
  • Higher log2 fold-changes in LPS-sensitive genes.
  • Greater divergence in transcriptomic profiles (PC1) between untreated and LPS-treated states.
• Gene Ontology:
  • Male-biased genes: Enriched for innate immunity (cytokine production, Toll-like receptor signaling).
  • Female-biased genes: Enriched for adaptive immunity (lymphocyte differentiation).
• Immunosenescence:
  • Older bats showed upregulation of innate immunity genes and downregulation of adaptive immunity genes.
  • Male Acceleration: Males exhibited steeper age-related slopes in immune gene expression. Specifically, modules related to B and T cell activation declined faster in males, while innate inflammation modules increased faster.
  • Leukocyte Influence: When the neutrophil-to-lymphocyte ratio (NLR) was included as a covariate, the magnitude of sex and age differences in gene expression decreased significantly, suggesting these differences are partly driven by shifts in circulating cell types.

C. Social Status

• Contrary to predictions based on primate studies, there were no large-scale differences in immune responses between harem and bachelor males.
• Only 41 genes differed between statuses, with no significant enrichment in specific biological processes, suggesting social adversity does not strongly drive immune variation in this species.

D. The Role of Mortality

• When ages were z-scaled independently for each sex (quantifying age relative to the species' lifespan distribution), the "male-biased immunosenescence" pattern disappeared. This suggests that the steeper immune decline in males is a reflection of their shorter lifespan and higher mortality rate (selective disappearance) rather than an intrinsic biological difference in aging rates per unit of time.

5. Significance

This study fundamentally shifts the perspective on bat immunology from a species-level "monolith" to an individual-level framework.

1. Life-History Trade-offs: It supports the hypothesis that the high energetic costs of male-male competition and polygyny in P. hastatus lead to resource reallocation away from somatic maintenance (including adaptive immunity), resulting in accelerated immunosenescence and male-biased mortality.
2. Context-Dependent Immunity: The lack of social status effects in bats, contrasting with primates, indicates that the link between social hierarchy and immunity is not universal and depends heavily on species-specific ecology and mating systems.
3. Longevity Mechanisms: The findings suggest that while bats generally possess unique immune adaptations for longevity, these mechanisms are modulated by individual life-history strategies. Understanding the interplay between sex, age, and social structure is essential for accurately modeling immune efficiency and the evolution of longevity in mammals.

In conclusion, the paper argues that future research on bat immunity must account for intraspecific variation in ecology and life history to fully understand the mechanisms underlying their disease tolerance and extended lifespans.