An inescapable consequence of sex in eukaryotes is the evolution of a biphasic life cycle with alternating diploid and haploid phases. The occurrence of selection during the haploid phase can have farreaching consequences for fundamental evolutionary processes including the rate of adaptation, the extent of inbreeding depression, and the load of deleterious mutations, as well as for applied research into fertilization technology. Although haploid selection is well established in plants, current dogma assumes that in animals, intact fertile sperm within a single ejaculate are equivalent at siring viable offspring. Using the zebrafish Danio rerio, we show that selection on phenotypic variation among intact fertile sperm within an ejaculate affects offspring fitness. Longer-lived sperm sired embryos with increased survival and a reduced number of apoptotic cells, and adult male offspring exhibited higher fitness. The effect on embryo viability was carried over into the second generation without further selection and was equally strong in both sexes. Sperm pools selected by motile phenotypes differed genetically at numerous sites throughout the genome. Our findings clearly link within-ejaculate variation in sperm phenotype to offspring fitness and sperm genotype in a vertebrate and have major implications for adaptive evolution.

It is generally believed that variation in sperm phenotype within a single ejaculate has no consequences for offspring performance, because sperm phenotypes are thought not to reflect sperm genotypes. We show that variation in individual sperm function within an ejaculate affects the performance of the resulting offspring in the Atlantic salmon Salmo salar. We experimentally manipulated the time between sperm activation and fertilization in order to select for sperm cohorts differing in longevity within single ejaculates of wild caught male salmon. We found that within-ejaculate variation in sperm longevity significantly affected offspring development and hence time until hatching. Whether these effects have a genetic or epigenetic basis needs to be further evaluated. However, our results provide experimental evidence for transgenerational effects of individual sperm function.

Summary1. Maternal age effects on life-history traits, including longevity, are widespread and can be seen as a manifestation of ageing. However, little is known about how maternal life span may influence the maternal age effect. At a given chronological age, a long-lived parent may be at a younger biological age than a short-lived parent and thus has a less severe parental age effect. However, earlier work using experimentally evolved short-and long-lived lines did not support this hypothesis. 2. We scored developmental time and longevity of 14 995 individual seed beetles, Callosobruchus maculatus derived from replicate short-lived and long-lived lines created via artificial selection on male life span. 3. Offspring from older mothers had shorter life span, which is consistent with most of the literature. 4. We found support for the hypothesis that detrimental maternal age effects evolve to be weaker under selection for long life span. However, this finding was only apparent in males, suggesting that maternal age affects male and female offspring differently. 5. These results suggest that sex-dependent parental age effects should be incorporated in the studies of longevity and ageing evolution and that selection on one sex can cause evolution of parental age effects in the other sex.

Males produce numerous sperm in a single ejaculate that greatly outnumber their potential egg targets. Recent studies found that phenotypic and genotypic variation among sperm in a single ejaculate of a male affects the fitness and performance of the resulting offspring. Specifically, within‐ejaculate sperm selection for sperm longevity increased the performance of the resulting offspring in several key life‐history traits in early life. Because increased early‐life reproductive performance often correlates with rapid ageing, it is possible that within‐ejaculate sperm selection increases early‐life fitness at the cost of accelerated senescence. Alternatively, within‐ejaculate sperm selection could improve offspring quality throughout the life cycle, including reduced age‐specific deterioration. We tested the two alternative hypotheses in an experimental setup using zebrafish Danio rerio . We found that within‐ejaculate sperm selection for sperm longevity reduced age‐specific deterioration of fecundity and offspring survival but had no effect on fertilization success in males. Remarkably, we found an opposing effect of within‐ejaculate sperm selection on female fecundity, where selection for sperm longevity resulted in increased early‐life performance followed by a slow decline, while females sired by unselected sperm started low but increased their fecundity with age. Intriguingly, within‐ejaculate sperm selection also reduced the age‐specific decline in fertilization success in females, suggesting that selection for sperm longevity improves at least some aspects of female reproductive ageing. These results demonstrate that within‐ejaculate variation in sperm phenotype contributes to individual variation in animal life histories in the two sexes and may have important implications for assisted fertilization programs in livestock and humans.

Genes of the major histocompatibility complex (MHC) are a likely target of mate choice because of their role in inbreeding avoidance and potential benefits for offspring immunocompetence. Evidence for female choice for complementary MHC alleles among competing males exists both for the pre- and the postmating stages. However, it remains unclear whether the latter may involve non-random fusion of gametes depending on gametic haplotypes resulting in transmission ratio distortion or non-random sequence divergence among fused gametes. We tested whether non-random gametic fusion of MHC-II haplotypes occurs in Atlantic salmon Salmo salar. We performed in vitro fertilizations that excluded interindividual sperm competition using a split family design with large clutch sample sizes to test for a possible role of the gametic haplotype in mate choice. We sequenced two MHC-II loci in 50 embryos per clutch to assess allelic frequencies and sequence divergence. We found no evidence for transmission ratio distortion at two linked MHC-II loci, nor for non-random gamete fusion with respect to MHC-II alleles. Our findings suggest that the gametic MHC-II haplotypes play no role in gamete association in Atlantic salmon and that earlier findings of MHC-based mate choice most likely reflect choice among diploid genotypes. We discuss possible explanations for these findings and how they differ from findings in mammals.

With thousands of loci identified by genome-wide association studies for complex traits, there is a need for in vivo model systems that can reliably and quickly infer the role of large numbers of candidate genes. CRISPR/Cas9-based functional screens in F0 zebrafish represent such a system. However, negative controls used so far - including scrambled guide RNAs (gRNAs), inactivated Cas9, and sham injections - do not elicit the same cellular and organismal responses as mutagenesis by CRISPR/Cas9, and may fuel biased conclusions. Here, we show that targetingkitafacilitates efficient optical pre-screening for successful mutagenesis, higher quality imaging data, and efficient classification of cases and controls. We identified and tested two gRNAs that targetkitawith similarly high mutagenic efficiency and effects on pigmentation, and are free from off-target effects or major effects on cardiometabolic traits. We propose several approaches that will result in valid, unbiased conclusions.

Introduction: Genome-wide association studies identified 100s of loci that are associated with the risk of common cardio-metabolic diseases (T2D and CVD). Here we functionally characterise 99 candidate genes for a role in relevant traits using zebrafish larvae. Methods: Fifty-six T2D and 46 CVD candidate genes (three overlap) were functionally characterised by targeting their zebrafish orthologs using CRISPR/Cas9. On day 9-11 post-fertilisation, we acquired optical sections of the pancreatic islet, liver and vasculature using semi-automated fluorescence microscopy, in 16,141 CRISPR/Cas9-edited zebrafish larvae, followed by image analysis using deep learning. Results: Perturbing all nine T2D genes with at least moderate prior evidence affects ≥1 T2D trait (i.e., beta cell mass, beta cell insulin expression, liver fat, and/or glucose content), while no consequence is observed for 22 genes without prior evidence of a role in T2D. For 12 genes with at most modest prior evidence, perturbation also affects at least 1 T2D trait. Perturbation of 23 genes prioritised for a role in CVD affects at least one vascular trait, while no consequence is observed for 15 non-prioritised genes. Effects of mutations in mice and/or humans are published for 14 of 23 genes, 78% of which show directionally consistent effects across zebrafish larvae and mammals. Of the 15 genes influencing liver fat upon CRISPR/Cas9 editing, mutations in 12 previously showed an effect on liver fat in mice and/or humans, with 75% congruence in direction of effect across species. Conclusion: Systematically characterising candidate genes for a role in image-based T2D, liver fat and CVD traits in CRISPR/Cas9-edited zebrafish larvae shows highly congruent results with mice and humans, and prioritises promising genes for further in-depth characterisation. Disclosure E. Mujica: None. H. Sun: None. M. Nyberg: Employee; Novo Nordisk A/S. D. Djordjevic: Employee; Novo Nordisk. S. Vienberg: Employee; Novo Nordisk A/S. A. L. Gloyn: Other Relationship; Genentech, Inc., Roche Pharmaceuticals. A. Larsson: None. A. Allalou: None. M. Den hoed: None. H. Zhang: None. A. Emmanouilidou: None. E. Mazzaferro: None. N. Cook: None. C. Metzendorf: None. G. Alavioon: None. M. Bandaru: None. A. Rottner: Employee; AstraZeneca. Funding Swedish Research Council (201901417); Swedish Heart and Lung Foundation (20200602, 20200781)

In most animals, males produce large numbers of sperm in each ejaculate, but only very few end up fertilising an egg. This bottleneck in sperm numbers from ejaculation to fertilisation offers an intuitive opportunity for selection to act and improve the fitness of the next generation. However, the general view that sperm phenotype is not linked to its haploid sperm genotype stalled further research into this idea until recently. Two studies in zebrafish Danio rerio now suggest that selection among sperm within the ejaculate of male may have far reaching consequences for the following generation(s).Selection for longer-lived sperm resulted in offspring that showed higher survival during embryo development and a reduced number of abnormally developed larvae, as well as increased reproductive success during adulthood. These effects have been linked to the haploid genotypes in the sperm. We here discuss the possible benefits of refined sperm selection based on sperm haplotypes in the use of Artificial Reproduction Technologies.Understanding the genetic processes occurring after meiosis until syngamy may provide insights that may help improve the existing methods and with that their success rates.