RNA interference has already proven itself to be a highly versatile molecular biology tool for understanding gene function in a limited number of insect species, but its widespread use in other species will be dependent on the development of easier methods of double-stranded RNA (dsRNA) delivery. This study demonstrates that RNA interference can be induced in the mosquito Aedes aegypti L. (Diptera: Culicidae) simply by soaking larvae in a solution of dsRNA for two hours. The mRNA transcripts for β-tubulin, chitin synthase-1 and -2, and heat shock protein 83 were reduced between 30 and 50% three days post-dsRNA treatment. The dsRNA was mixed with a visible dye to identify those individuals that fed on the dsRNA, and based on an absence of RNA interference in those individuals that contained no dye within their guts, the primary route of entry of dsRNA is likely through the gut epithelium. RNA interference was systemic in the insects, inducing measurable knock down of gene expression in tissues beyond the gut. Silencing of the β-tubulin and chitin synthase-1 genes resulted in reduced growth and/or mortality of the larvae, demonstrating the utility of dsRNA as a potential mosquito larvicide. Silencing of chitin synthase-2 did not induce mortality in the larvae, and silencing of heat shock protein 83 only induced mortality in the insects if they were subsequently subjected to a heat stress. Drosophila melanogaster Meigen (Diptera: Drosophilidae) larvae were also soaked in dsRNA designed to specifically target either their own β-tubulin gene, or that of A. aegypti, and significant mortality was only seen in larvae treated with dsRNA targeting their own gene, which suggests that dsRNA pesticides could be designed to be species-limited.

Chronic inflammation contributes to a wide range of human diseases, and environments in infancy and childhood are important determinants of inflammatory phenotypes. The underlying biological mechanisms connecting early environments with the regulation of inflammation in adulthood are not known, but epigenetic processes are plausible candidates. We tested the hypothesis that patterns of DNA methylation (DNAm) in inflammatory genes in young adulthood would be predicted by early life nutritional, microbial, and psychosocial exposures previously associated with levels of inflammation. Data come from a population-based longitudinal birth cohort study in metropolitan Cebu, the Philippines, and DNAm was characterized in whole blood samples from 494 participants (age 20–22 y). Analyses focused on probes in 114 target genes involved in the regulation of inflammation, and we identified 10 sites across nine genes where the level of DNAm was significantly predicted by the following variables: household socioeconomic status in childhood, extended absence of a parent in childhood, exposure to animal feces in infancy, birth in the dry season, or duration of exclusive breastfeeding. To evaluate the biological significance of these sites, we tested for associations with a panel of inflammatory biomarkers measured in plasma obtained at the same age as DNAm assessment. Three sites predicted elevated inflammation, and one site predicted lower inflammation, consistent with the interpretation that levels of DNAm at these sites are functionally relevant. This pattern of results points toward DNAm as a potentially important biological mechanism through which developmental environments shape inflammatory phenotypes across the life course.

Evolutionary theory predicts that reproduction entails costs that detract from somatic maintenance, accelerating biological aging. Despite support from studies in human and non-human animals, mechanisms linking ‘costs of reproduction’ (CoR) to aging are poorly understood. Human pregnancy is characterized by major alterations in metabolic regulation, oxidative stress, and immune cell proliferation. We hypothesized that these adaptations could accelerate blood-derived cellular aging. To test this hypothesis, we examined gravidity in relation to telomere length (TL, n = 821) and DNA-methylation age (DNAmAge, n = 397) in a cohort of young (20–22 year-old) Filipino women. Age-corrected TL and accelerated DNAmAge both predict age-related morbidity and mortality, and provide markers of mitotic and non-mitotic cellular aging, respectively. Consistent with theoretical predictions, TL decreased (p = 0.031) and DNAmAge increased (p = 0.007) with gravidity, a relationship that was not contingent upon resource availability. Neither biomarker was associated with subsequent fertility (both p > 0.3), broadly consistent with a causal effect of gravidity on cellular aging. Our findings provide evidence that reproduction in women carries costs in the form of accelerated aging through two independent cellular pathways.

Objectives: Socioeconomic status (SES) is a powerful determinant of health, but the underlying biological mechanisms are poorly understood. This study investigates whether levels of DNA methylation at CpG sites across the genome are associated with SES in a cohort of young adults in the Philippines.Methods: DNA methylation was assayed with the Illumina HumanMethylation450 Bead Chip, in leukocytes from 489 participants in the Cebu Longitudinal Health and Nutrition Survey (mean age = 20.9 years). SES was measured in infancy/childhood and adulthood, and was based on composite measures of income, assets, and education. Genome-wide analysis of variable probes identified CpG sites significantly associated with SES after adjustment for multiple comparisons. Functional enrichment analysis was used to identify biological pathways associated with these sites.Results: A total of 2,546 CpG sites, across 1,537 annotated genes, were differentially methylated in association with SES. In comparison with high SES, low SES was associated with increased methylation at 1,777 sites, and decreased methylation at 769 sites. Functional enrichment analysis identified over-representation of biological pathways related to immune function, skeletal development, and development of the nervous system.Conclusions: Socioeconomic status predicts DNA methylation at a large number of CpG sites across the genome. The scope of these associations is commensurate with the wide range of biological systems and health outcomes that are shaped by SES, and these findings suggest that DNA methylation may play an important role. K E Y W O R D S epigenetics, health disparities, human growth and development

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All humans age, but how we age—and how fast—differs considerably from person to person. This deviation between apparent age and chronological age is often referred to as “biological age” (BA) and until recently robust tools for studying BA have been scarce. “Epigenetic clocks” are starting to change this. Epigenetic clocks use predictable changes in the epigenome, usually DNA methylation, to estimate chronological age with unprecedented accuracy. More importantly, deviations between epigenetic age and chronological age predict a broad range of health outcomes and mortality risks better than chronological age alone. Thus, epigenetic clocks appear to capture fundamental molecular processes tied to BA and can serve as powerful tools for studying health, development, and aging across the lifespan. In this article, I review epigenetic clocks, especially as they relate to key theoretical and applied issues in human biology. I first provide an overview of how epigenetic clocks are constructed and what we know about them. I then discuss emerging applications of particular relevance to human biologists—those related to reproduction, life‐history, stress, and the environment. I conclude with an overview of the methods necessary for implementing epigenetic clocks, including considerations of study design, sample collection, and technical considerations for processing and interpreting epigenetic clocks. The goal of this review is to highlight some of the ways that epigenetic clocks can inform questions in human biology, and vice versa, and to provide human biologists with the foundational knowledge necessary to successfully incorporate epigenetic clocks into their research.

We validated a radioimmunoassay-based method quantifying fecal glucocorticoid metabolites (FGMs) from captive male and female Richardson’s ground squirrels Urocitellus richardsonii. Blood samples were drawn to explore the correlation between plasma cortisol and FGM concentrations. We also injected groups of squirrels with normal saline (CTL; control), adre-nocorticotropic hormone (ACTH; stimulating adrenal activity), or dexamethasone (DEX; suppressing adrenal activity). Potential correlations between stress and behaviour were explored through quantification of fecal pellet production and the intervention necessary to elicit defecation, as well as the behaviour of subjects in the context of handling. Changes in plasma cortisol concentration between capture (baseline), and following handling (stress-induced) were also quantified for free-living squirrels. While glucocorticoid concentrations recovered from feces during our captive-animal study were not well correlated with plasma cortisol concentrations, and uncorrelated with defecation or behaviour, FGM concentrations did reflect the activation of the hypothalamic-pituitary-adrenal (HPA) axis. FGM concentrations increased significantly during initial captivity, but declined to baseline level as individuals acclimated to the novel environment. Injection of subjects with ACTH increased FGMs above baseline, confirming activation of the HPA axis. Plasma cortisol concentrations increased significantly with induced stress, indicating that capture and handling activated the glucocorticoid stress response even among previously handled, free-living subjects. Our findings validate a non-invasive tool that will afford new insight into the physiological processes underlying social, reproductive and antipredator behaviour of Richardson’s ground squirrels.

Partnered fathers often have lower testosterone than single non-parents, which is theorized to relate to elevated testosterone (T) facilitating competitive behaviors and lower T contributing to nurturing. Cultural- and individual-factors moderate the expression of such psychobiological profiles. Less is known about genetic variation's role in individual psychobiological responses to partnering and fathering, particularly as related to T. We examined the exon 1 CAG (polyglutamine) repeat (CAGn) within the androgen receptor (AR) gene. AR CAGn shapes T's effects after it binds to AR by affecting AR transcriptional activity. Thus, this polymorphism is a strong candidate to influence individual-level profiles of "androgenicity." While males with a highly androgenic profile are expected to engage in a more competitive-oriented life history strategy, low androgenic men are at increased risk of depression, which could lead to similar outcomes for certain familial dynamics, such as marriage stability and parenting. Here, in a large longitudinal study of Filipino men (n=683), we found that men who had high androgenicity (elevated T and shorter CAGn) or low androgenicity (lower T and longer CAGn) showed elevated likelihood of relationship instability over the 4.5-year study period and were also more likely be relatively uninvolved with childcare as fathers. We did not find that CAGn moderated men's T responses to the fatherhood transition. In total, our results provide evidence for invested fathering and relationship stability at intermediate levels of androgenicity and help inform our understanding of variation in male reproductive strategies and the individual hormonal and genetic differences that underlie it.