The evolution of Mullerian or Batesian mimicry depends on the relative unpalatability of participating taxa, yet the mechanistic basis of such unpalatability remains poorly explored in many systems. Unpalatability in the Corydoradinae catfishes, known for their striking mimetic diversity, is underpinned by sharp, lockable spines, armoured bodies and the production of venoms/toxins. Here, we assess the contribution of the axillary gland, a structure at the base of the pectoral spine, to toxicity in two Corydoradinae genera, Corydoras and Hoplisoma. Using a brine shrimp cytotoxicity assay, we demonstrate that axillary gland extracts are significantly more toxic than muscle extracts in both genera, but toxicity does not differ significantly between genera. Transcriptomic analyses identified 539 candidate toxin genes upregulated in axillary gland tissue, relative to scute tissue, containing signal peptides and had predicted toxin functions based on amino acid sequences. Notably, candidate genes include domains typical of piscine venoms, such as lectins and peptidase S1. Although significant differences in gene expression were detected between genera in candidate toxins, log-fold changes were small, and predicted toxin potency was not significantly different between genera. Together, our findings indicate that axillary gland toxins are likely to contribute to unpalatability in Corydoradinae catfishes and provide support for Mullerian mimicry in this system. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Science Research Council, BB/R017174/1, BB/T00871/1 Fisheries Society of the British Isles, FSBI-RG22-192

Environmental factors affect not only paternal condition but may translate into the following generations where sperm-mediated small RNAs (sRNAs) can contribute to the transmission of paternal effects. sRNAs play a key role in the male germ line in genome maintenance and repair, and particularly in response to environmental stress and the resulting increase in transposable element (TE) activity. Here, we investigated how the social environment (high competition, low competition) of male zebrafish Danio rerio affects sRNAs in sperm and how these are linked to gene expression and TE activity in their offspring. In a first experiment, we collected sperm samples after exposing males to each social environment for 2 weeks to test for differentially expressed sperm micro- (miRNA) and piwi-interacting RNAs (piRNA). In a separate experiment, we performed in vitro fertilisations after one 2-week period using a split-clutch design to control for maternal effects and collected embryos at 24 h to test for differentially expressed genes and TEs. We developed new computational prediction tools to link sperm sRNAs with differentially expressed TEs and genes in the embryos. Our results support the idea that the molecular stress response in the male germ line has significant down-stream effects on the molecular pathways, and we provide a direct link between sRNAs, TEs and gene expression.

Reduced insulin/IGF-1 signalling (IIS) robustly extends lifespan and enhances somatic stress resistance across taxa, yet its consequences for germline genome integrity remain unclear. Here we combine multigenerational mutation accumulation with whole-genome sequencing in C. elegans to test whether adulthood-only IIS downregulation can simultaneously promote somatic maintenance and limit germline mutational burden. We reduced IIS by adult-onset daf-2 RNAi in wild-type and heritable RNAi-deficient ( hrde-1 ) backgrounds, allowing either spontaneous or UV-induced germline mutations to accumulate over multiple generations. In wild-type animals, reduced IIS lowered germline single-nucleotide mutation rates by up to ∼50% and prevented the UV-induced elevation in mutation rate, without detectable costs to fecundity or lineage persistence. By contrast, in hrde-1 mutants the same intervention increased both point mutations and transposable-element–driven insertions under UV exposure, accelerating lineage extinction. Thus, the genome-protective effect of reduced IIS critically requires the germline nuclear Argonaute HRDE-1, which mediates small-RNA–guided epigenetic silencing. Functional annotation of germline variants revealed enrichment in pathways linked to development, cellular maintenance and conserved longevity regulators, including IIS and mTOR, and identified high-impact mutations in genes with human orthologs implicated in neurodegeneration and cancer. Our findings show that IIS can coordinate somatic and germline maintenance in concert, rather than in competition, through an HRDE-1–dependent epigenetic pathway. This work positions nutrient-sensing IIS as a central regulator of germline genome stability and suggests that IIS downregulation can reduce germline mutation load while extending lifespan, with broad implications for biogerontology and evolutionary biology.

Gum Arabic (GA) (Acacia senegal var. senegal) is an edible tree exudate and dietary fibre shown to improve health in humans and animals. We tested the ramifications of GAon organismal health across the microbiota-gut-brain axis by supplementing female and male zebrafish (Danio rerio) with two concentrations(6% or 60%) of GA (Acacia senegal var. senegal) for two weeks. We assessed the effects on the gut microbiome composition, intestinal and brain metabolic profiles, reproductive fitness, locomotion, and brain gene expression. GA supplementation induced a relative decrease in Proteobacteria and a relative increase in Fusobacteria, with a rise in the beneficial genus Cetobacterium. In the GA-supplemented fish, we detected increased intestinal glucose metabolism, evidenced by reduced glucose retention levels. Additionally, high levels of acetate were detected in the brain. Interestingly, the gene cart1, involved in appetite and hunger control, was significantly downregulated in female brains only. Consistently, we detected increased locomotion in GA-supplemented fish compared to Control fish. Interestingly, GA supplementation had a negative effect on female reproductive fitness and a positive effect on male reproductive fitness. .Our results emphasise the significance of evaluating the impact of dietary fibre at a systemic level to develop relevant nutritional guidelines that consider the different nutritional requirements of each sex.

Inbreeding and the associated increase in homozygosity and potential accumulation of deleterious alleles may reduce fitness in a process known as inbreeding depression. Mechanisms to mitigate reproduction between close relatives, ranging from pre-mating mate choice to post-mating gamete selection, have evolved across taxa. In external fertilisers like Atlantic salmon (Salmo salar), where females have limited control over paternity, mechanisms of inbreeding avoidance can be expected to evolve at the gamete level. Philopatric Atlantic salmon may run the risk of breeding between relatives, particularly in small populations, and frequent escapes from aquaculture settings are augmenting the chances of adults from the same sibling cohort overlapping in the wild, raising potential ecological and sustainability concerns. Moreover, the presence of inbreeding avoidance mechanisms between full siblings in externally fertilising fish is currently untested. This study tested post-mating inbreeding avoidance mechanisms in domesticated Atlantic salmon. In a paired breeding design, we compared sperm motility parameters in sibling and non-sibling ovarian fluid, and assessed fertilisation and hatching success, growth rate and paternity in sperm competition trials between sibling and non-sibling males. Sperm activated in ovarian fluid of sibling females showed lower values of motility-related parameters and led to an average of 18% reduction in fertilisation rates in the resulting crosses. Furthermore, offspring from sibling crosses were smaller before the onset of sexual maturation. However, we found no difference in survival rates between sibling and non-sibling cross offspring. Besides, when sperm from sibling and non-sibling males were competing simultaneously for the same egg batch, we found no influence of this on paternity. Our findings reveal the presence of post-mating inbreeding avoidance at the gamete level in Atlantic salmon, but its effects are limited in competitive scenarios. Our results have implications for salmonid conservation and aquaculture, where small natural or closed domestic strains may both display a degree of inbreeding. Mating between escaped domestic and wild salmonids could favour admixed over wild or feral crosses if an inbreeding avoidance mechanism is present; although this remains to be tested in more outbred crosses.

The inheritance of small RNAs (sRNAs) is taxonomically widespread. Changing environments alter the production and presence of sRNAs in the germline, and this can in theory either increase offspring phenotypic variance as an evolutionary bet-hedging strategy or elicit predictive and adaptive phenotypic responses that increase offspring fitness. Nevertheless, the putative role of sRNA inheritance systems in adaptive evolution is still debated and it is currently unclear how selection acts on sRNAs. We outline two adaptive sRNA inheritance strategies - specialist and generalist - and discuss non-adaptive alternatives and the evolutionary implications of different strategies. Our review suggests that the role of natural selection in sRNA inheritance has been significantly overlooked, potentially leading to misinterpretations of the causal agents and the evolutionary implications of sRNA inheritance.

Intergenerational and transgenerational fitness effects can shape evolutionary processes. Theoretically, however, intergenerational and transgenerational effects can trade-off with each other with profound consequences for evolutionary processes. Here we show that beneficial intergenerational effects that increase offspring fitness can result in detrimental transgenerational effects that decrease great-grand offspring fitness. We combined theoretical modelling and experimental approaches to investigate multigenerational fitness trade-offs induced by larval starvation in Caenorhabditis elegans. We demonstrate that larval starvation triggers a cascading effect: starved individuals suffered marked fitness losses, their direct offspring enjoyed fitness gains in both starvation and ad libitum environments, but great-grand-offspring paid fitness costs. Demographic simulation models revealed that genotypes exploiting this short-term intergenerational advantage outcompete rival genotypes despite the deferred transgenerational debt. Our findings demonstrate that adaptive intergenerational gains can be intrinsically linked to maladaptive transgenerational outcomes, challenging the assumption that transgenerational effects are inherently beneficial and highlighting the role of multigenerational trade-offs in evolution.

BACKGROUND:Piwi-interacting RNAs (piRNA)s are non-coding small RNAs that post-transcriptionally affect gene expression and regulation. Through complementary seed region binding with transposable elements (TEs), piRNAs protect the genome from transposition. A tool to link piRNAs with complementary TE targets will improve our understanding of the role of piRNAs in genome maintenance and gene regulation. Existing tools such as TEsmall can process sRNA-seq datasets to produce differentially expressed piRNAs, and piRScan developed for nematodes can link piRNAs and TEs but it requires knowledge about the target region of interest and works backwards. RESULTS:We developed FishPi to predict the pairings between piRNA and TEs for available genomes from zebrafish, medaka and tilapia, with full user customisation of parameters including orientation of piRNA, mismatches in the piRNA seed binding to TE and scored output lists of piRNA-TE matches. FishPi works with individual piRNAs or a list of piRNA sequences in fasta format. The software focuses on the piRNA-TE seed region and analyses reference TEs for piRNA complementarity. TE type is examined, counted and stored to a dictionary, with genomic loci recorded. Any updates to piRNA-TE binding rules can easily be incorporated by changing the seed-region options in the graphic user-interface. FishPi provides a graphic interface using tkinter for the user to input piRNA sequences to generate comprehensive reports on piRNA-TE interactions. FishPi can easily be adapted to genomes from other species and taxa opening the interpretation of piRNA functionality to a wide community. CONCLUSIONS:Users will gain insight into genome mobility and FishPi will help further our understanding of the biological role of piRNAs and their interaction with TEs in a similar way that public databases have improved the access to and the understanding of the role of small RNAs.

Environmental fluctuations influence heritable phenotypes through complex molecular mechanisms. In zebrafish (Danio rerio ), the interplay between temperature variation, transposable element (TE) activity, and small RNA (sRNA) regulation in the germline remains underexplored. Here we exposed adult zebrafish to elevated temperatures and performed integrated genomic, transcriptomic, and sRNA profiling of testes and ovaries. We observed sex-specific responses: ovaries exhibited enhanced protein-processing pathways and robust miRNA and piRNA-mediated TE suppression, whereas testes showed immune-related signatures, reduced piRNA output, and increased de novo TE insertions preferentially within introns. These coordinated changes link environmental stress to genomic and epigenetic modulation, suggesting mechanisms by which populations may rapidly adapt to fluctuating conditions. Our findings provide insight into sex-biased germline resilience and highlight the role of TE dynamics and sRNA pathways in mediating thermal adaptation. ### Competing Interest Statement The authors have declared no competing interest. * DE/DEG : differentially expressed /differentially expressed gene miRNA : microRNA piRNA : piwi interacting RNA sRNA : small RNA TE : transposable element tRNA : transfer RNA European Research Council, https://ror.org/0472cxd90, SELECTHAPLOID - 101001341 Natural Environment Research Council, NE/S011188/1

While the definition of germ cell fate has been extensively studied in model organisms, evolutionary innovations and mechanistic novelties may remain hidden in understudied systems. The phenomenon of programmed DNA elimination allows germ cells to acquire germline-restricted genes, offering a novel paradigm of germ cell specificity. In passerine birds, the germline-restricted chromosome (GRC) is eliminated from somatic cells in early embryogenesis, yet the role and consequences of its maintenance in the germ cells remain poorly understood. Here, using the zebra finch Taeniopygia guttata as a model, we combined RNA-seq and Spatial Transcriptomics to construct a high-resolution spatiotemporal expression map to understand the role of the GRC across germ cell development. We found a GRC-linked integrin-BMP signaling in maturing oocytes and tfeb GRC upregulation at blastoderm embryos, suggesting the involvement of the GRC in oocyte maturation and germ cell determination. We also identified developmental specialization of GRC-linked gene expression relative to their paralogs on the autosomes and sex chromosomes, revealing a gene repertoire which promotes germline stemness and germline/soma distinction. Together, the passerine GRC constitutes a unique system that manifests germ cell complexity, whilst allowing pinpointing the effects on gene expression that may elucidate vertebrate germ cell fate.

Dietary restriction (DR) extends lifespan in animals and plants, but its evolutionary causes are elusive. Adaptive hypotheses posit that DR extends lifespan because organisms reallocate resources from reproduction to survival (‘disposable soma’) or recycle cellular waste to maximize either their immediate reproduction (‘nutrient recycling’) or survival (‘clean cupboards’). We developed an experimental paradigm that tricks Caenorhabditis elegans nematodes into increasing their reproductive effort under DR via food odour, thus allowing us to test these hypotheses. We found that experimentally increased reproduction under DR does not affect immediate or long-term survival benefits compared to DR animals that did not reproduce, thus refuting all three adaptive hypotheses. Our data suggest that a large part of suppressed fertility under DR is a result of organisms refraining from producing offspring in a poor environment. We developed a model based on Hamiltonian forces of selection to show that lifespan extension under DR evolves because DR suppresses fertility, directly increasing selection against mortality in DR environment. Our analytical approach suggests that DR-driven lifespan extension can evolve under a broader range of conditions not previously anticipated, such as a relaxed need for physiological or genetic trade-offs. Instead, we show how reduced survival on plentiful food can evolve via mutation accumulation. ### Competing Interest Statement The authors have declared no competing interest.

A new subpopulation of polar bears (Ursus maritimus) was recently discovered in the South-East of Greenland. This isolated colony inhabits a warmer climate zone, akin to the predicted future environments of polar bears with vastly reduced sea ice habitats, rendering this population of bears particularly important. Over two-thirds of polar bears will be extinct by 2050 with total extinction predicted by the end of this century, therefore understanding possible mechanisms of adaptation via genomic analyses and preservation are critical. Transposable elements (TEs) are mobile elements that may play a role in an adaptive response to environmental challenges. We analysed transcriptome data from polar bear sub-populations in cooler North-East (NEG) and warmer South-East Greenland (SEG) to compare TE activity between the two populations and its correlation with temperature and associated changes in gene expression. We identified activity hotspots in the genome of regions with significantly differentially expressed TEs. LINE family TEs were the most abundant, and most differentially expressed and divergent in the SEG population compared to reference TEs. We report a significant shift in TE activity and age, with younger more abundant TEs in the SEG populations. Differentially expressed genes in SEG populations were linked to Foxo signalling, ageing and metabolic pathways. Our results provide insights into how a genomic response at the TE level may allow the SEG subpopulations to adapt and survive to climate change and provides a useful resource for conservation in polar bears.

One of the key processes that forms the basis of fertilisation is the tight interaction between sperm and egg. Both sperm and egg proteomes are known to evolve and diverge rapidly even between closely related species. Understanding the sperm proteome therefore provides key insights into the proteins that underpin the mechanisms involved during fertilisation and the fusion between sperm and egg, and how they can differ across individuals of the same species. Despite being a commonly used model organism for reproductive research, little is currently understood about the zebrafish Danio rerio sperm proteome. We performed nanoLC-MS/MS proteomics analysis after off-line sample fractionation with six pooled samples containing sperm from ten males each. We confidently identified 5410 proteins, from which a total of 3900 GeneIDs were generated leading to 1720 Gene Ontology terms.

The evolution of separate sexes from cosexuality requires at least two mutations: a feminizing allele to cause female development and a masculinizing allele to cause male development. Classically, the double mutant is assumed to be sterile, which leads to two-factor sex determination where male and female sex chromosomes differ at two loci. However, several species appear to have one-factor sex determination where sexual development depends on variation at a single locus. We show that one-factor sex determination evolves when the double mutant develops as a male or a female. The feminizing allele fixes when the double mutant is male, and the masculinizing allele fixes when the double mutant is female. The other locus then gives XY or ZW sex determination based on dominance: for example, a dominant masculinizer becomes a Y chromosome. Although the resulting sex determination system differs, the conditions required for feminizers and masculinizers to spread are the same as in classical models, with the important difference that the two alleles do not need to be linked. Thus, we reveal alternative pathways for the evolution of sex determination and discuss how they can be distinguished using new data on the genetics of sex determination.

As the threat of climate change and associated heatwaves grows, we need to understand how natural populations will respond. Inter-generational non-genetic inheritance may play a key role in rapid adaptation, but whether such mechanisms are truly adaptive and sufficient to protect wild populations is unclear. The contribution of paternal effects in particular is not fully understood, even though the male reproductive system may be highly sensitive to heatwaves. We used the zebrafish Danio rerio to investigate the effects of heatwaves on male fertility and assess potential adaptive benefits to their offspring in a number of large-scale heatwave experiments. Heatwave conditions had negative effects on male fertility by reducing gamete quality and fertilisation success, and we found indications of an adaptive effect on hatching in offspring produced by heatwave-exposed males. Our findings highlight the importance of including male and female fertility when determining species ability to cope with extreme conditions and suggest that parental effects provide limited adaptive benefits.

Competition among pollen or sperm (gametic selection) can cause evolution. Mating systems shape the intensity of gametic selection by determining the competitors involved, which can in turn cause the mating system itself to evolve. We model the bidirectional relationship between gametic selection and mating systems, focusing on variation in female mating frequency (monandry-polyandry) and self-fertilisation (selfing-outcrossing). First, we find that monandry and selfing both reduce the efficiency of gametic selection in removing deleterious alleles. This means that selfing can increase mutation load, in contrast to cases without gametic selection where selfing purges deleterious mutations and decreases mutation load. Second, we explore how mating systems evolve via their effect on gametic selection. By manipulating gametic selection, polyandry can evolve to increase the fitness of the offspring produced. However, this indirect advantage of post-copulatory sexual selection is weak and is likely to be overwhelmed by any direct fitness effects of mating systems. Nevertheless, gametic selection can be potentially decisive for selfing evolution because it significantly reduces inbreeding depression, which favours selfing. Thus, the presence of gametic selection could be a key factor driving selfing evolution.

Dietary restriction in the form of fasting is a putative key to a healthier and longer life, but these benefits may come at a trade-off with reproductive fitness and may affect the following generation(s). The potential inter- and transgenerational effects of long-term fasting and starvation are particularly poorly understood in vertebrates when they originate from the paternal line. We utilised the externally fertilising zebrafish amenable to a split-egg clutch design to explore the male-specific effects of fasting/starvation on fertility and fitness of offspring independently of maternal contribution. Eighteen days of fasting resulted in reduced fertility in exposed males. While average offspring survival was not affected, we detected increased larval growth rate in F1 offspring from starved males and more malformed embryos at 24 h post-fertilisation in F2 offspring produced by F1 offspring from starved males. Comparing the transcriptomes of F1 embryos sired by starved and fed fathers revealed robust and reproducible increased expression of muscle composition genes but lower expression of lipid metabolism and lysosome genes in embryos from starved fathers. A large proportion of these genes showed enrichment in the yolk syncytial layer suggesting gene regulatory responses associated with metabolism of nutrients through paternal effects on extra-embryonic tissues which are loaded with maternal factors. We compared the embryo transcriptomes to published adult transcriptome datasets and found comparable repressive effects of starvation on metabolism-associated genes. These similarities suggest a physiologically relevant, directed and potentially adaptive response transmitted by the father, independently from the offspring's nutritional state, which was defined by the mother.

Dietary fibres (DFs) constitute a wide range of heterogeneous compounds that resist digestion and have beneficial effects on general health. Gum Arabic (GA) is a tree exudate consisting of 90% arabinogalactan, a polymer of arabinose and galactose sugars with prebiotic properties. As a dietary fibre, GA improves renal function, metabolism, and immune response in humans and animals. However, the underlying mechanisms leading to these health benefits are poorly understood. We supplemented female and male zebrafish ( Danio rerio ) with two concentrations of GA (6% and 60%) for two weeks. We assessed the effects of GA supplementation on the gut microbiome composition, intestinal and brain metabolic profiles, reproductive fitness, and brain gene expression. We found that GA supplementation resulted in changes to the gut microbiome with a relative increase in Fusobacteria and a relative decrease in Proteobacteria where the beneficial genus Cetobacterium was significantly more abundant after supplementation. GA supplementation increased acetate levels, particularly in the brain, causing a decreased expression of cart1 in the brain of female zebrafish. While GA supplementation increased overall activity in male and female fish, reproductive fitness was negatively affected by GA supplementation in females. Our results suggest that while GA supplementation may have positive effects on metabolic rate and overall activity, it may come at a trade-off with reproductive fitness. ### Competing Interest Statement The authors have declared no competing interest.

Males produce millions of sperm in a single ejaculate, but only a single sperm fertilises the egg. Fertilisation is the perfect opportunity for natural selection where sperm of poor quality are eliminated, and the fittest sperm sire the offspring. However, the biomarkers of the fittest sperm are currently elusive. Here, we selected human sperm pools for fitness within ejaculates of healthy donors using in vitro assays and identify phenotypic, genomic and proteomic biomarkers of fitter sperm. Fitter sperm showed prolonged motility and incrsed morphological normality and DNA condensation, and their genomes and proteomes differed from less fit sperm. The genes diverging between sperm pools are involved in neurodevelopmental processes, cell signalling and cell proliferation and are linked to heritable diseases including neurodevelopmental disorders and cancers. The genomic signatures of haploid selection in human sperm strongly overlapped in function with the genomic signature in zebrafish sperm suggesting that the sperm functions under haploid selection are highly conserved. The five top peptides downregulated in fitter sperm across all donors are known markers for inflammation, immune function and cancer-related cell proliferation. Selecting for high-performance long-lived sperm prior to fertilisation may therefore help prevent ageing-related heritable diseases in the offspring later in life. ### Competing Interest Statement The authors have declared no competing interest.

The environment gametes perform in just before fertilization is increasingly recognized to affect offspring fitness, yet the contributions of male and female gametes and their adaptive significance remain largely unexplored. Here, we investigated gametic thermal plasticity and its effects on hatching success and embryo performance in Atlantic salmon (Salmo salar). Eggs and sperm were incubated overnight at 2°C or 8°C, temperatures within the optimal thermal range of this species. Crosses between warm- and cold-incubated gametes were compared using a full-factorial design, with half of each clutch reared in cold temperatures and the other in warm temperatures. This allowed disentangling single-sex interaction effects when pre-fertilization temperature of gametes mismatched embryonic conditions. Pre-fertilization temperature influenced hatch timing and synchrony, and matching sperm and embryo temperatures resulted in earlier hatching. Warm incubation benefited eggs but harmed sperm, reducing the hatching success and, overall, gametic thermal plasticity did not enhance offspring fitness, indicating vulnerability to thermal changes. We highlight the sensitivity of male gametes to higher temperatures, and that gamete acclimation may not effectively buffer against deleterious effects of thermal fluctuations. From an applied angle, we propose the differential storage of male and female gametes as a tool to enhance sustainability within the hatcheries.