Adaptation of organisms to extreme environments requires dramatic phenotypic changes. Studying these changes can elucidate mechanisms underlying phenotypic differences in the context of both evolution and human disease. The Mexican tetra, Astyanax mexicanus, is a powerful model of extreme adaptation over a short evolutionary time scale. This fish species includes surface- and cave-dwelling ecotypes, with cavefish displaying many adaptations to subterranean life, including behavioral changes such as sleep loss, increased appetite, and reduced aggression. Unraveling the mechanisms underlying these changes has been challenging, presumably because they are complex traits that required coordinated changes across multiple cell types to evolve. Here, we present a spatially integrated comparative cell atlas of whole adult brains of surface and cavefish. After establishing the molecular signatures of 35 cell types, we show that cave colonization drove canalized regulatory changes to gene expression across diverse cell types. Cavefish brains show shifts in cell-type composition compared to their surface counterparts, as well as complex regulatory changes to pathways governing hypoxia response and circadian rhythm. Microglia in the cavefish brain underwent extensive transcriptional remodelling, including changes in senescence and AMPK pathways. Further, cell-cell communication analysis identified a cave-enriched ligand-receptor communication pattern centered on signals sent from glial cells to diverse populations of neurons. This atlas identifies genetic changes associated with neural and behavioral evolution and provides a resource for mechanistic studies examining brain evolution.

Cortisol is a key hormone involved in stress responses, and its regulation can vary across populations experiencing different environmental pressures. While short term changes in certain environmental factors can result in immediate changes in cortisol levels, it remains unclear whether variable and fluctuating environments affect lifetime stress responses of individuals. In this study, we estimated lifetime-experienced stress in cave- and surface-dwelling populations of Astyanax mexicanus. The advantage of this model is that different populations are confronted with varied dynamics of environmental change, making it an ideal study system to investigate lifetime stress responses upon environmental fluctuations. To estimate lifetime-experienced stress, we measured cortisol levels in the scales of A. mexicanus for the first time using an ELISA assay. We studied six populations from six different habitats (four cave and two surface habitats) that vary in their dynamics of abiotic factors. We analysed scale cortisol levels across two years to examine temporal changes and individual variability within and between populations. Our results show that increased fluctuations in abiotic parameters are associated with an increased cortisol level in scales and, in addition, an increased inter-individual variability within a population irrespective of cave or surface habitat. These findings highlight the influence of varying ecological conditions on lifetime-experienced stress in A. mexicanus, providing insight into potential adaptive mechanisms underlying stress regulation in extreme environments. ### Competing Interest Statement The authors have declared no competing interest.

The Mexican tetra A. mexicanus, with its phenotypically distinct cave- and surface-dwelling conspecific morphs, has provided broad insight into adaptive phenotypes. Recent research highlights the intricate interplay between the brain and immune system, leading us to hypothesize that the unique environmental pressures of cave life have reshaped intercellular communication within the immune system of the brain. To investigate this, we generated single-nuclei and spatial transcriptome atlases of the adult brains of surface and cavefish to explore immune system adaptation. Analysis of 116,000 nuclei allowed us to annotate 35 cell types, including of immune cell clusters showing mixed lineage characteristics. Subsequent reclustering of immune cells identified 19 transcriptionally distinct populations with multiple classifications of microglia (n = 4), macrophages (n = 2), and T cells (n = 3) seen. The evaluation of cell type compositional shifts using a Bayesian proportionality test, showed significant change for a population of microglia and T cells, in both higher numbers observed in the cave adapted fish. Interestingly, preliminary evaluations of other A. mexicanus organ-systems also show immune cell type specific compositional differences. This catalog of immune cell changes in the genetically adapted brain enhances our understanding of how gene regulatory networks mediate the integration of immune and nervous system functions. NIH R24OD030214 Veterinary and Comparative Immunology (VET)

Genetic variation fuels evolution, and the release of cryptic variation is key for adaptation. The heat shock protein 90 (HSP90) has been proposed to act as an evolutionary capacitor by revealing such hidden variation under stress. However, this idea remains debated, as the genetic basis of HSP90-regulated traits is often unknown, and many observed phenotypes are deleterious. Here, we show in Tribolium castaneum that HSP90 shapes evolution by unmasking a hidden trait providing enhanced fitness under specific conditions. Using RNA interference and chemical inhibition, we consistently reveal a reduced-eye phenotype that persists in descendant lines across generations without continued HSP90 disruption. Under constant light, reduced-eye beetles had higher reproductive success and greater trait penetrance than normal-eyed siblings, suggesting a selective advantage. Whole-genome sequencing and functional analysis identify the transcription factor atonal (ato) as the underlying gene. These results provide the first direct genetic link between an HSP90-buffered trait and context-dependent fitness benefits in animals, highlighting a potential mechanism by which cryptic variation contributes to adaptation.

Immune memory has long been thought to be restricted to the adaptive immune system of vertebrates. However, several lines of evidence have changed our understanding of immune memory and have shattered the strict separation between innate and adaptive immunity. In vertebrates, a form of innate immunity that is called ‘trained immunity’ has been intensively studied for over a decade. For more than two decades, studies in plants and an increasing number of invertebrate taxa have clearly demonstrated that these organisms also possess immune memory, despite the absence of an adaptive immune system. These phenomena are mostly known as ‘immune priming’. The mechanistic underpinnings of immune priming vary across taxa and may or may not partially include the epigenetic and metabolic mechanisms involved in trained immunity. Here, we offer an evolutionary perspective on immune priming, uniquely integrating key aspects across plants and invertebrates for the first time. As a basis, we provide a conceptual clarification regarding the terms trained immunity and immune priming and give a brief overview of these phenomena across taxa. We then analyze which processes of immune priming share potentially evolutionary conserved epigenetic and metabolic processes with trained immunity and explore signaling processes involved in immune priming. We discuss the aspect of specificity as one of the key defining criteria for immune memory and incorporate the potential role of soil and gut microbiota for acquiring innate immune memory in plants and invertebrates. Finally, we argue that immune priming has enormous potential for application beyond the medical field when involving the protection against parasites and pathogens in agriculture and aquaculture.

To date, the intricate cellular and molecular interplay between hosts and parasites is well described for a limited number of model systems. Recent methodological advances now allow deep insights in non-model symbioses, including such that ultimately appear barely antagonistic and may entail intense reciprocal manipulations. We applied single cell RNA-sequencing to blood samples of a wild, long-lived avian host, the Common Buzzard (Buteo buteo), infected with the malaria-like parasite Leucocytozoon toddi. We identified host cell types and their respective molecular toolkits involved in both, host immunity and parasite virulence. Comparing blood cell-type specific transcriptomic profiles between Buzzards with low and high parasitaemia, we gain insights into the parasitic immune evasion and potential modulation of the host immune response. The response of immune cells was overall low indicating immunosuppression under high parasitaemia, despite the reportedly low pathogenicity of the parasite, likely a result of long-lasting coevolution. Our study illuminates in rare detail a benign host-parasite interaction and bridges a large phylogenetic and symbiotic gap in the evolutionary gradient from parasitism to mutualism. The cell-type specific markers which we identify allow a more complete understanding of the evolutionary forces shaping host immunity. ### Competing Interest Statement The authors have declared no competing interest.

Glutathione transferases from the omega class are notable for their roles in redox regulation and cellular stress response. In this study, we conducted a comprehensive functional characterization of GST-44, an omega-class glutathione S-transferase (GSTO), in Caenorhabditis elegans, focusing on its role in cellular defense mechanisms against stress. Biochemical analysis revealed GSTO-specific enzymatic activities of recombinant GST-44, including dehydroascorbate reductase, thioltransferase, and arsenate reductase activities. Using transgenic GFP reporter strains, we identified predominant expression of GST-44 in the intestine and excretory H-cell, with significant upregulation observed under diverse stress conditions. Induction of GST-44 was particularly pronounced in the intestine in response to pathogen-, oxidative-, and endoplasmic reticulum stress. Notably, under arsenic stress, the expression of gst-44 was significantly upregulated in the excretory system of the worm, underscoring its critical role in mediating arsenic detoxification. Moreover, we demonstrated the induction of GST-44 using dimethyl fumarate, a highly specific mammalian Nrf-2 activator. The upregulation of GST-44 during arsenic stress was dependent not only on the oxidative stress response transcription factor SKN-1/Nrf2 but also on PHA-4. The deletion mutant strain gst-44(tm6133) exhibited reduced stress resistance and a shortened lifespan, with a highly diminished survival rate under arsenic stress compared to other CRISPR-generated C. elegans GSTO deletion mutants. Our findings highlight the essential role of GST-44 in mediating arsenic detoxification, as well as in stress adaptation and defense mechanisms in C. elegans.

BACKGROUND:Research on forms of memory in innate immune systems has recently gained momentum with the study of trained immunity in vertebrates and immune priming in invertebrates. Immune priming is an evolutionary ancient process that confers protection against previously encountered pathogens. However, despite the existence of immune priming across many invertebrate taxa, evolution and mechanisms of immune priming are still not well understood. Moreover, it is unclear how natural pathogens might elicit immune priming in their hosts. RESULTS:Here we combine RNA sequencing with transmission electron microscopy to investigate the dynamic processes during priming in the gut of a well-established model for oral immune priming, consisting of the host Tribolium castaneum and its natural pathogen Bacillus thuringiensis tenebrionis (Btt). We show that priming with specific, pathogen-derived virulence-relevant factors induces gut damage in T. castaneum larvae, triggering an early physiological stress response and upregulation of a distinct set of immune genes. This response diminishes over time yet enables the gut to upregulate genes known to interfere with Btt virulence when later exposed to infectious Btt spores. CONCLUSIONS:Our findings demonstrate that pathogen-derived factors inducing gut damage and stress responses prime gut tissue to provide more efficient protection against infection. These insights deepen our understanding of the mechanisms driving innate immune memory, which likely evolved as an adaptive response to natural pathogens.

The ecological and genetic changes that underlie the evolution of host-microbe interactions remain elusive, primarily due to challenges in disentangling the variables that alter microbiome composition. To understand the impact of host habitat, host genetics, and evolutionary history on microbial community structure, we examined gut microbiomes of river- and three cave-adapted morphotypes of the Mexican tetra, Astyanax mexicanus, in their natural environments and under controlled laboratory conditions. Field-collected samples were dominated by very few taxa and showed considerable interindividual variation. We found that lab-reared fish exhibited increased microbiome richness and distinct composition compared to their wild counterparts, underscoring the significant influence of habitat. Most notably, however, we found that morphotypes reared on the same diet throughout life developed distinct microbiomes suggesting that genetic loci resulting from cavefish evolution shape microbiome composition. We observed stable differences in Fusobacteriota abundance between morphotypes and demonstrated that this could be used as a trait for quantitative trait loci mapping to uncover the genetic basis of microbial community structure.

Sleep is a complex and conserved biological process that affects several body functions and behaviors. Evidence suggests that there is a reciprocal interaction between sleep and immunity. For instance, fragmented sleep can increase the probability of parasitic infections and reduce the ability to fight infections. Moreover, viral and bacterial infections alter the sleep patterns of infected individuals. However, the effects of macro-parasitic infections on sleep remain largely unknown, and measuring sleep in non-model organisms remains challenging. In this study, we investigated whether macro-parasite infections could alter sleep-like behavior of their hosts. We experimentally infected three-spined sticklebacks (Gasterosteus aculeatus), a freshwater fish, with the tapeworm Schistocephalus solidus and used a hidden Markov model to characterize sleep-like behavior in sticklebacks. One to four days after parasite exposure, infected fish showed no difference in sleep-like behavior compared with non-exposed fish, and fish that were exposed-but-not-infected only showed a slight reduction in sleep-like behavior during daytime. Twenty-nine to 32 days after exposure, infected fish showed more sleep-like behavior than control fish, while exposed-but-not-infected fish showed overall less sleep-like behavior. Using brain transcriptomics, we identified immune- and sleep-associated genes that potentially underlie the observed behavioral changes. These results provide insights into the complex association between macro-parasite infection, immunity, and sleep in fish and may thus contribute to a better understanding of reciprocal interactions between sleep and immunity.

The ecological and genetic changes that underlie evolution of host-microbe interactions remain elusive, primarily due to challenges in disentangling the variables that alter microbiome composition. To understand the impact of host habitat, host genetics and evolutionary history on microbial community structure, we examined gut microbiomes of river- and three cave-adapted morphotypes of the Mexican tetra, Astyanax mexicanus , in their natural environments and under controlled laboratory conditions. We found that lab-reared fish exhibited increased microbiome richness and distinct composition compared to their wild counterparts, underscoring the significant influence of habitat. Most notably, however, we found that morphotypes reared on the same diet throughout life developed distinct microbiomes suggesting that genetic loci resulting from cavefish adaptation shape microbiome composition. We observed stable differences in Fusobacteriota abundance between morphotypes and demonstrate that this could be used as a trait for quantitative trait loci mapping to uncover the genetic basis of microbial community structure.

The ecological and genetic changes that underlie evolution of host-microbe interactions remain elusive, primarily due to challenges in disentangling the variables that alter microbiome composition. To understand the impact of host habitat, host genetics and evolutionary history on microbial community structure, we examined gut microbiomes of river- and three cave-adapted morphotypes of the Mexican tetra, Astyanax mexicanus, in their natural environments and under controlled laboratory conditions. We found that lab-reared fish exhibited increased microbiome richness and distinct composition compared to their wild counterparts, underscoring the significant influence of habitat. Most notably, however, we found that morphotypes reared on the same diet throughout life developed distinct microbiomes suggesting that genetic loci resulting from cavefish adaptation shape microbiome composition. We observed stable differences in Fusobacteriota abundance between morphotypes and demonstrate that this could be used as a trait for quantitative trait loci mapping to uncover the genetic basis of microbial community structure.

The microorganisms that reside in animal intestines are influenced by the habitat and genetics of the host. The mechanisms that govern host-microbe interactions are subject to natural selection as they are heritable and impact host fitness. In this study, we use the Mexican tetra, Astyanax mexicanus, to investigate how rapidly evolution can shape host-driven intestinal microbiome composition. A. mexicanus is a species of fish consisting of river-dwelling surface fish and multiple eyeless cave-dwelling cavefish that diverged from surface fish less than 200 thousand years ago. Surface fish and cavefish have been bred in laboratories for generations to study the genetic and developmental basis of cavefish traits like eye and pigment loss, reduced aggression, adiposity, and insulin resistance. We used 16S rRNA sequencing to compare the intestinal microbiome of surface fish and cavefish that were collected in the wild, to the same populations of fish that were raised from embryos under identical conditions in the lab. We found that microbial species richness and composition are altered both by habitat (wild versus lab) and by morphotype identity (surface versus cave). Reared under identical conditions, surface fish develop a microbiome that is significantly different from more distantly related cavefish (Pachón and Tinaja), but not a more closely related cavefish (Molino), suggesting microbial community relationships recapitulate the phylogeny of the host. The most striking difference in microbiome composition between morphotypes is near absence of Fusobacteriota of the genus Cetobacterium in Pachón cavefish. Low abundance of Cetobacterium in zebrafish is associated with impaired glucose homeostasis and increased sensitivity to pathogens, mimicking traits observed in Pachón cavefish. We found that Fusobacteriota abundance is a stable trait that could be used for quantitative trait loci mapping in A. mexicanus to investigate the genetic basis of microbiome composition. Our study shows that evolution in an underground cave can rapidly alter host genetics to influence microbiome composition in ways that could impact the metabolism of the host.

Physical inactivity is a scourge to human health, promoting metabolic disease and muscle wasting. Interestingly, multiple ecological niches have relaxed investment into physical activity, providing an evolutionary perspective into the effect of adaptive physical inactivity on tissue homeostasis. One such example, the Mexican cavefish Astyanax mexicanus, has lost moderate-to-vigorous activity following cave colonization, reaching basal swim speeds ~3.7-fold slower than their river-dwelling counterpart. This change in behavior is accompanied by a marked shift in body composition, decreasing total muscle mass and increasing fat mass. This shift persisted at the single muscle fiber level via increased lipid and sugar accumulation at the expense of myofibrillar volume. Transcriptomic analysis of laboratory-reared and wild-caught cavefish indicated that this shift is driven by increased expression of pparγ —the master regulator of adipogenesis—with a simultaneous decrease in fast myosin heavy chain expression. Ex vivo and in vivo analysis confirmed that these investment strategies come with a functional trade-off, decreasing cavefish muscle fiber shortening velocity, time to maximal force, and ultimately maximal swimming speed. Despite this, cavefish displayed a striking degree of muscular endurance, reaching maximal swim speeds ~3.5-fold faster than their basal swim speeds. Multi-omic analysis suggested metabolic reprogramming, specifically phosphorylation of Pgm1-Threonine 19, as a key component enhancing cavefish glycogen metabolism and sustained muscle contraction. Collectively, we reveal broad skeletal muscle changes following cave colonization, displaying an adaptive skeletal muscle phenotype reminiscent to mammalian disuse and high-fat models while simultaneously maintaining a unique capacity for sustained muscle contraction via enhanced glycogen metabolism.

Cis-regulatory changes are key drivers of adaptative evolution. However, their contribution to the metabolic adaptation of organisms is not well understood. Here, we used a unique vertebrate model, Astyanax mexicanus-different morphotypes of which survive in nutrient-rich surface and nutrient-deprived cave waters-to uncover gene regulatory networks underlying metabolic adaptation. We performed genome-wide epigenetic profiling in the liver tissues of Astyanax and found that many of the identified cis-regulatory elements (CREs) have genetically diverged and have differential chromatin features between surface and cave morphotypes, while retaining remarkably similar regulatory signatures between independently derived cave populations. One such CRE in the hpdb gene harbors a genomic deletion in cavefish that abolishes IRF2 repressor binding and derepresses enhancer activity in reporter assays. Selection of this mutation in multiple independent cave populations supports its importance in cave adaptation, and provides novel molecular insights into the evolutionary trade-off between loss of pigmentation and adaptation to food-deprived caves.

Cell lines have become an integral resource and tool for conducting biological experiments ever since the Hela cell line was first developed (Scherer et al. in J Exp Med 97:695–710, 1953). They not only allow detailed investigation of molecular pathways but are faster and more cost-effective than most in vivo approaches. The last decade saw many emerging model systems strengthening basic science research. However, lack of genetic and molecular tools in these newer systems pose many obstacles. Astyanax mexicanus is proving to be an interesting new model system for understanding metabolic adaptation. To further enhance the utility of this system, we developed liver-derived cell lines from both surface-dwelling and cave-dwelling morphotypes. In this study, we provide detailed methodology of the derivation process along with comprehensive biochemical and molecular characterization of the cell lines, which reflect key metabolic traits of cavefish adaptation. We anticipate these cell lines to become a useful resource for the Astyanax community as well as researchers investigating fish biology, comparative physiology, and metabolism.

Physical inactivity – specifically the lack of moderate-to-vigorous activity – is a scourge to human health, promoting metabolic disease and muscle wasting. Interestingly, multiple ecological niches have relaxed investment into physical activity, providing unique evolutionary insight into adaptive physical inactivity. The Mexican cavefish Astyanax mexicanus lost moderate-to-vigorous activity following cave colonization, reaching basal swim speeds ~3-fold slower than their river-dwelling counterpart. We found that this was accompanied by a marked shift in body composition, decreasing muscle mass by 30% and increasing fat mass by 40%. This shift persisted at the single muscle fiber level via increased lipid and sugar accumulation at the expense of myofibrillar volume. Transcriptomic analysis of laboratory-reared and wild-caught cavefish indicated this shift in investment is driven by increased expression of pparγ – the master regulator of adipogenesis – with a simultaneous decrease in fast myosin heavy chain expression. Ex vivo and in vivo analysis confirmed these investment strategies come with a functional trade-off, decreasing cavefish muscle fiber shortening velocity, time to maximal force, and ultimately maximal swimming velocity. Despite this, cavefish displayed a striking degree of muscular endurance, reaching maximal swim speeds ~3.5-fold faster than their basal swim speeds. Multi-omics analysis indicated metabolic reprogramming, specifically increased phosphoglucomutase-1 abundance, phosphorylation, and activity, as contributing mechanisms enhancing cavefish glycogen utilization under metabolically strenuous conditions. Collectively, we reveal broad skeletal muscle reprogramming following cave colonization, displaying an adaptive skeletal muscle phenotype reminiscent to mammalian disuse and high-fat models while simultaneously maintaining a unique capacity for sustained muscle contraction under fatiguing conditions. Significance The evolutionary consequence of decreased physical activity upon skeletal muscle physiology remains unexplored. Using the Mexican cavefish, we find loss of moderate-to-vigorous swimming following cave colonization has resulted in broad shifts in skeletal muscle investment – away from muscle mass and instead toward fat and sugar accumulation – ultimately decreasing muscle fiber twitch kinetics. Surprisingly though, cavefish possessed marked muscular endurance, reaching maximal swimming speeds rivaling their river-dwelling counterpart. Multi-omics analysis revealed carbohydrate metabolic reprogramming as a contributing component, most notably elevated abundance and phosphorylation of the glycogenolytic enzyme Phosphoglucomutase-1 – a likely adaptation to cave-specific hypoxia. These findings emphasize the impact multiple selective pressures have on skeletal muscle physiology, providing the first evolutionary insight into skeletal muscle adaptation following decreased activity.

Nutrient availability varies seasonally and spatially in the wild. The resulting nutrient limitation or restricted access to nutrients pose a major challenge for every organism. While many animals, such as hibernating animals, evolved strategies to overcome periods of nutrient scarcity, the cellular mechanisms of these strategies are poorly understood. Cave environments represent an extreme example of nutrient deprived environments since the lack of sunlight and therefore primary energy production drastically diminishes the nutrient availability. Here, we used Astyanax mexicanus , which includes river-dwelling surface fish and cave adapted cavefish populations to study the genetic adaptation to nutrient limitations. We show that cavefish populations store large amounts of fat in different body regions when fed ad libitum in the lab. We found higher expression of lipogenesis genes in cavefish livers when fed the same amount of food as surface fish, suggesting an improved ability of cavefish to use lipogenesis to convert available energy into triglycerides for storage into adipose tissue. Moreover, the lipid metabolism regulator, Peroxisome proliferator-activated receptor γ (Pparγ), is upregulated at both transcript and protein levels in cavefish livers. Chromatin Immunoprecipitation sequencing (ChIP seq) showed that Pparγ binds cavefish promoter regions of genes to a higher extent than surface fish. Finally, we identified two possible regulatory mechanisms of Pparγ in cavefish: higher amounts of ligands of the nuclear receptor, and nonsense mutations in per2 , a known repressor of Pparγ. Taken together, our study reveals that upregulated Pparγ promotes higher levels of lipogenesis in the liver and contributes to higher body fat accumulation in cavefish populations, an important adaptation to nutrient limited environments.

Insights from organisms, which have evolved natural strategies for promoting survivability under extreme environmental pressures, may help guide future research into novel approaches for enhancing human longevity. The cave-adapted Mexican tetra, Astyanax mexicanus, has attracted interest as a model system for metabolic resilience, a term we use to denote the property of maintaining health and longevity under conditions that would be highly deleterious in other organisms (Figure 1). Cave-dwelling populations of Mexican tetra exhibit elevated blood glucose, insulin resistance and hypertrophic visceral adipocytes compared to surface-dwelling counterparts. However, cavefish appear to avoid pathologies typically associated with these conditions, such as accumulation of advanced-glycation-end-products (AGEs) and chronic tissue inflammation. The metabolic strategies underlying the resilience properties of A. mexicanus cavefish, and how they relate to environmental challenges of the cave environment, are poorly understood. Here, we provide an untargeted metabolomics study of long- and short-term fasting in two A. mexicanus cave populations and one surface population. We find that, although the metabolome of cavefish bears many similarities with pathological conditions such as metabolic syndrome, cavefish also exhibit features not commonly associated with a pathological condition, and in some cases considered indicative of an overall robust metabolic condition. These include a reduction in cholesteryl esters and intermediates of protein glycation, and an increase in antioxidants and metabolites associated with hypoxia and longevity. This work suggests that certain metabolic features associated with human pathologies are either not intrinsically harmful, or can be counteracted by reciprocal adaptations. We provide a transparent pipeline for reproducing our analysis and a Shiny app for other researchers to explore and visualize our dataset.

Intergenerational effects from fathers to offspring are increasingly reported in diverse organisms, but the underlying mechanisms are often unknown. Paternal trans-generational immune priming (TGIP) was demonstrated in the red flour beetle Tribolium castaneum: non-infectious bacterial exposure (priming) of fathers protects their offspring against an infectious challenge for at least two generations. Here we studied a potential role of the Dnmt2 gene (renamed as Trdmt1 in humans), which encodes a highly conserved enzyme that methylates specific cytosines of a set of tRNAs and has previously been reported to be involved in intergenerational epigenetic inheritance in mice. We first studied gene expression and found that Dnmt2 was expressed throughout life, with high expression in testes. Knockdown of Dnmt2 in fathers slowed down offspring larval development and increased mortality of the adult offspring upon bacterial infection. However, the observed effects were independent of the paternal priming treatment. In conclusion, our results point towards a role of Dnmt2 for paternal effects, while elucidation of the mechanisms behind paternal immune priming needs further studies.