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International audience; Climate warming is considered to be among the most serious of anthropogenic stresses to the environment, because it not only has direct effects on biodiversity, but it also exacerbates the harmful effects of other human-mediated threats. The associated consequences are potentially severe, particularly in terms of threats to species preservation, as well as in the preservation of an array of ecosystem services provided by biodiversity. Among the most affected groups of animals are insects—central components of many ecosystems—for which climate change has pervasive effects from individuals to communities. In this contribution to the scientists' warning series, we summarize the effect of the gradual global surface temperature increase on insects, in terms of physiology, behavior, phenology, distribution, and species interactions, as well as the effect of increased frequency and duration of extreme events such as hot and cold spells, fires, droughts, and floods on these parameters. We warn that, if no action is taken to better understand and reduce the action of climate change on insects, we will drastically reduce our ability to build a sustainable future based on healthy, functional ecosystems. We discuss perspectives on relevant ways to conserve insects in the face of climate change, and we offer several key recommendations on management approaches that can be adopted, on policies that should be pursued, and on the involvement of the general public in the protection effort.

Why is polyandry such a common mating behaviour when it exposes females to a range of significant fitness costs? Here, we investigated whether polyandry protects females against reduced male fertility caused by thermal stress from heatwave conditions. Sperm production and function are vulnerable to heat, and heatwave conditions are forecast to increase as our climate warms, so we examined these effects on female reproduction and mating behaviour in the flour beetle, Tribolium castaneum, a promiscuous ectotherm model in which fertility is damaged by environmental warming. We tested whether polyandrous matings, or polyandrous sperm stores, protect females against reduced male fertility caused by heatwave conditions, and whether females flexibly adjust their remating behaviour to enable fertility rescue. We found that polyandry protected females against reduced male fertility: monogamous matings with males exposed to heatwave conditions halved female offspring production, but opportunities to mate with five of these males allowed normal female reproductive output. By contrast with this fertility improvement following polyandrous mating opportunities, there was no protective benefit for females already carrying sperm stores from multiple males, which suffered similar heatwave damage within the female tract as monogamous sperm stores. Importantly, female polyandry was flexible to male condition, with females showing greater motivation to remate with new males if their previous mate had been damaged by heatwave exposure, enabling a rapid reproductive rescue. Our results reveal that flexible polyandry enables females to rescue their fertility when male reproductive function is compromised by heatwave conditions, a phenomenon that may become more prevalent under climate change.

Gamete-level sexual selection of externally fertilising species is usually achieved by modifying sperm behaviour with mechanisms that alter the chemical environment in which gametes perform. In fish, this can be accomplished through the ovarian fluid, a substance released with the eggs at spawning. While the biochemical effects of ovarian fluid in relation to sperm energetics have been investigated, the influence of the physical environment in which sperm compete remains poorly explored. Our objective was therefore to gain insights on the physical structure of this fluid and potential impacts on reproduction. Using soft-matter physics approaches of steady-state and oscillatory viscosity measurements, we subjected wild Atlantic salmon ovarian fluids to variable shear stresses and frequencies resembling those exerted by sperm swimming through the fluid near eggs. We show that this fluid, which in its relaxed state is a gel-like substance, displays a non-Newtonian viscoelastic and shear-thinning profile, where the viscosity decreases with increasing shear rates. We concurrently find that this fluid obeys the Cox–Merz rule below 7.6 Hz and infringes it above this level, thus indicating a shear-thickening phase where viscosity increases provided it is probed gently enough. This suggests the presence of a unique frequency-dependent structural network with relevant implications for sperm energetics and fertilisation dynamics. This article has an associated ECR Spotlight interview with Marco Graziano.

Flour beetles of the genus Tribolium have been utilised as informative study systems for over a century and contributed to major advances across many fields. This review serves to highlight the significant historical contribution that Tribolium study systems have made to the fields of ecology and evolution, and to promote their use as contemporary research models. We review the broad range of studies employing Tribolium to make significant advances in ecology and evolution. We show that research using Tribolium beetles has contributed a substantial amount to evolutionary and ecological understanding, especially in the fields of population dynamics, reproduction and sexual selection, population and quantitative genetics, and behaviour, physiology and life history. We propose a number of future research opportunities using Tribolium, with particular focus on how their amenability to forward and reverse genetic manipulation may provide a valuable complement to other insect models.

Male genitals are highly divergent in animals with internal fertilization. Most studies attempting to explain this diversity have focused on testing the major hypotheses of genital evolution (the lock‐and‐key, pleiotropy, and sexual selection hypotheses), and quantifying the form of selection targeting male genitals has played an important role in this endeavor. However, we currently know far less about selection targeting female genitals or how male and female genitals interact during mating. Here, we use formal selection analysis to show that genital size and shape is subject to strong multivariate stabilizing sexual selection in both sexes of the red flour beetle, Tribolium castaneum. Moreover, we show significant sexual selection on the covariance between the sexes for specific aspects of genital shape suggesting that male and female genitalia also interact to determine the successful transfer of a spermatophore during mating. Our work therefore highlights the important role that both male and female genital morphologies play in determining mating success and that these effects can occur independently, as well as through their interaction. Moreover, it cautions against the overly simplistic view that the sexual selection targeting genital morphology will always be directional in form and restricted primarily to males.

Sperm length unexpectedly varies more than 3,000-fold across species, posing new questions for anisogamy theory and understanding the different forces shaping evolution of the male gamete.

Polyandry, when females mate with more than one male, is theorised to play an important role in successful colonisation of new habitats. In addition to possible benefits from sexual selection, even mild polyandry could facilitate colonisation by protecting against inbreeding and reducing the costs of mating with incompatible or infertile males. Here, we measure the importance of mild polyandry for population viability and reproductive fitness following experimental founder events into a higher‐temperature regime. Using colonisation experiments with the model beetle Tribolium castaneum, in which females can produce offspring for up to 140 days following a single mating, we founded more than 100 replicate populations using single females that had been given the opportunity to mate with either one or two males and then tracked their subsequent population dynamics. Following population viability and fitness across 10 generations, we found that extinction rates were significantly lower in populations founded by females given polyandrous opportunities to mate with two males (9%) compared to populations founded by monogamous females (34%). In addition, populations founded by females that had been provided with opportunities to store sperm from two different males showed double the median productivity following colonisation compared to monogamous‐founded populations. Notably, we identified short‐term and longer‐term benefits to post‐colonisation populations from double‐mating, with results suggesting that polyandry acts to both protect against mating with incompatible males through the founder event, and reduce inbreeding depression as the colonisation proceeds for 10 generations. Our results therefore show that even mild polyandry provides both reproductive and genetic benefits for colonising populations., We performed an evolution experiment to test the hypothesis that polyandry can enable populations to successfully colonise new environments. We found that populations founded by polyandrous females were less likely to go extinct and grew larger in size than those founded by monogamous females.

Earth's biodiversity is undergoing mass extinction due to anthropogenic compounding of environmental, demographic and genetic stresses. These different stresses can trap populations within a reinforcing feedback loop known as the extinction vortex, in which synergistic pressures build upon one another through time, driving down population viability. Sexual selection, the widespread evolutionary force arising from competition, choice and reproductive variance within animal mating patterns could have vital consequences for population viability and the extinction vortex: (a) if sexual selection reinforces natural selection to fix ‘good genes’ and purge ‘bad genes’, then mating patterns encouraging competition and choice may help protect populations from extinction; (b) by contrast, if mating patterns create load through evolutionary or ecological conflict, then population viability could be further reduced by sexual selection. We test between these opposing theories using replicate populations of the model insect Tribolium castaneum exposed to over 10 years of experimental evolution under monogamous versus polyandrous mating patterns. After a 95‐generation history of divergence in sexual selection, we compared fitness and extinction of monogamous versus polyandrous populations through an experimental extinction vortex comprising 15 generations of cycling environmental and genetic stresses. Results showed that lineages from monogamous evolutionary backgrounds, with limited opportunities for sexual selection, showed rapid declines in fitness and complete extinction through the vortex. By contrast, fitness of populations from the history of polyandry, with stronger opportunities for sexual selection, declined slowly, with 60% of populations surviving by the study end. The three vortex stresses of (a) nutritional deprivation, (b) thermal stress and (c) genetic bottlenecking had similar impacts on fitness declines and extinction risk, with an overall sigmoid decline in survival through time. We therefore reveal sexual selection as an important force behind lineages facing extinction threats, identifying the relevance of natural mating patterns for conservation management., Global Change Biology, 26 (8), ISSN:1354-1013, ISSN:1365-2486

Rising and more variable global temperatures pose a challenge for biodiversity, with reproduction and fertility being especially sensitive to heat. Here, we assessed the potential for thermal adaptation in sperm and egg function using Tribolium flour beetles, a warm-temperate-tropical insect model. Following temperature increases through adult development, we found opposing gamete responses, with males producing shorter sperm and females laying larger eggs. Importantly, this gamete phenotypic plasticity was adaptive: thermal translocation experiments showed that both sperm and eggs produced in warmer conditions had superior reproductive performance in warmer environments, and vice versa for cooler production conditions and reproductive environments. In warmer environments, gamete plasticity enabled males to double their reproductive success, and females could increase offspring production by one-third. Our results reveal exciting potential for sensitive but vital traits within reproduction to handle increasing and more variable thermal regimes in the natural environment.

Endangered species face a huge array of challenges, including the negative consequences of individuals having to breed with close genetic relatives. But just how costly is inbreeding in small populations? New research from an endangered bird species suggests that considering inbreeding could be crucial for conservation programmes.

Rensch's rule proposes a universal allometric scaling phenomenon across species where sexual size dimorphism (SSD) has evolved: in taxa with male-biased dimorphism, degree of SSD should increase with overall body size, and in taxa with female-biased dimorphism, degree of SSD should decrease with increasing average body size. Rensch's rule appears to hold widely across taxa where SSD is male-biased, but not consistently when SSD is female-biased. Furthermore, studies addressing this question within species are rare, so it remains unclear whether this rule applies at the intraspecific level. We assess body size and SSD within Tribolium castaneum (Herbst), a species where females are larger than males, using 21 populations derived from separate locations across the world, and maintained in isolated laboratory culture for at least 20 years. Body size, and hence SSD patterns, are highly susceptible to variations in temperature, diet quality and other environmental factors. Crucially, here we nullify interference of such confounds as all populations were maintained under identical conditions (similar densities, standard diet and exposed to identical temperature, relative humidity and photoperiod). We measured thirty beetles of each sex for all populations, and found body size variation across populations, and (as expected) female-biased SSD in all populations. We test whether Rensch's rule holds for our populations, but find isometry, i.e. no allometry for SSD. Our results thus show that Rensch's rule does not hold across populations within this species. Our intraspecific test matches previous interspecific studies showing that Rensch's rule fails in species with female-biased SSD.

Climate change is affecting biodiversity, but proximate drivers remain poorly understood. Here, we examine how experimental heatwaves impact on reproduction in an insect system. Male sensitivity to heat is recognised in endotherms, but ectotherms have received limited attention, despite comprising most of biodiversity and being more influenced by temperature variation. Using a flour beetle model system, we find that heatwave conditions (5 to 7 °C above optimum for 5 days) damaged male, but not female, reproduction. Heatwaves reduce male fertility and sperm competitiveness, and successive heatwaves almost sterilise males. Heatwaves reduce sperm production, viability, and migration through the female. Inseminated sperm in female storage are also damaged by heatwaves. Finally, we discover transgenerational impacts, with reduced reproductive potential and lifespan of offspring when fathered by males, or sperm, that had experienced heatwaves. This male reproductive damage under heatwave conditions provides one potential driver behind biodiversity declines and contractions through global warming., Animal physiology, including reproduction, could respond to climate change in complex ways. Here, the authors use experiments with an insect model system to show that simulated heatwaves harm male reproductive potential by reducing sperm number and viability, an effect which persisted into the next generation

The existence of widespread male same-sex sexual behaviour (SSB) is puzzling: why does evolution allow costly homosexual activity to exist, when reproductive fitness is primarily achieved through heterosexual matings? Here, we used experimental evolution to understand why SSB occurs in the flour beetle Tribolium castaneum. By varying the adult operational sex ratio across 82–106 generations, we created divergent evolutionary regimes that selected for or against SSB depending upon its function. Male-biased (90:10 M:F) regimes generated strong selection on males from intrasexual competition, and demanded improved ability to locate and identify female mates. By contrast, Female-biased regimes (10:90 M:F) generated weak male–male competition, and relaxed selection on mate-searching abilities in males. If male SSB functions through sexually selected male–male competition, it should be more evident within Male-biased regimes, where reproductive competition is nine times greater, than in the Female-biased regimes. By contrast, if SSB exists due to inaccurate mate choice, it should be reduced in Male-biased regimes, where males experience stronger selection for improved mate finding and discrimination abilities than in the Female-biased regime, where most potential mating targets are female. Following these divergent evolutionary regimes, we measured male engagement in SSB through choice experiments simultaneously presenting female and male mating targets. Males from both regimes showed similar overall levels of mating activity. However, there were significant differences in levels of SSB between the two regimes: males that evolved through male-biased operational sex ratios located, mounted and mated more frequently with the female targets. By contrast, males from female-biased selection histories mated less frequently with females, exhibiting almost random choice between male and female targets in their first mating attempt. Following experimental evolution, we therefore conclude that SSB does not function through sexually selected male–male competition, but instead occurs because males fail to perfectly discriminate females as mates., Animal Behaviour, 139, ISSN:0003-3472, ISSN:1095-8282

Because of divergent selection acting on males and females arising from different life-history strategies, polyandry can be expected to promote sexual dimorphism of investment into immune function. In previous work we have established the existence of such divergence within populations where males and females are exposed to varying degrees of polyandry. We here test whether the removal of sexual selection via enforced monogamy generates males and females that have similar levels of investment in immune function. To test this prediction experimentally, we measured differences between the sexes in a key immune measurement (phenoloxidase (PO) activity) and resistance to the microsporidian Paranosema whitei in Tribolium castaneum lines that evolved under monogamous (sexual selection absent) vs polyandrous (sexual selection present) mating systems. At generation 49, all selected lines were simultaneously assessed for PO activity and resistance to their natural parasite P. whitei after two generations of relaxed selection. We found that the polyandrous regime was associated with a clear dimorphism in immune function: females had significantly higher PO activities than males in these lines. In contrast, there was no such difference between the sexes in the lines evolving under the monogamous regime. Survival in the infection experiment did not differ between mating systems or sexes. Removing sexual selection via enforced monogamy thus seems to erase intersexual differences in immunity investment. We suggest that higher PO activities in females that have evolved under sexual selection might be driven by the increased risk of infections and/or injuries associated with exposure to multiple males.

It is the differences between sperm and eggs that fundamentally underpin the differences between the sexes within reproduction. For males, it is theorized that widespread sperm competition leads to selection for investment in sperm numbers, achieved by minimizing sperm size within limited resources for spermatogenesis in the testis. Here, we empirically examine how sperm competition shapes sperm size, after more than 77 generations of experimental selection of replicate lines under either high or low sperm competition intensities in the promiscuous flour beetle Tribolium castaneum. After this experimental evolution, populations had diverged significantly in their sperm competitiveness, with sperm in ejaculates from males evolving under high sperm competition intensities gaining 20% greater paternity than sperm in ejaculates from males that had evolved under low sperm competition intensity. Males did not change their relative investment into sperm production following this experimental evolution, showing no difference in testis sizes between high and low intensity regimes. However, the more competitive males from high sperm competition intensity regimes had evolved significantly longer sperm and, across six independently selected lines, there was a significant association between the degree of divergence in sperm length and average sperm competitiveness. To determine whether such sperm elongation is costly, we used dietary restriction experiments, and revealed that protein-restricted males produced significantly shorter sperm. Our findings therefore demonstrate that sperm competition intensity can exert positive directional selection on sperm size, despite this being a costly reproductive trait.

Adaptations at the gamete level (a) evolve quickly, (b) appear sensitive to inbreeding and outbreeding and (c) have important influences on potential to reproduce. We apply this understanding to problems posed by escaped farm salmon and measure their potential to reproduce in the wild. Farm Atlantic salmon (Salmo salar) are a threat to biodiversity, because they escape in large numbers and can introgress, dilute or disrupt locally adapted wild gene pools. Experiments at the whole fish level have found farm reproductive potential to be significant, but inferior compared to wild adults, especially for males. Here, we assess reproductive performance at the gamete level through detailed in vitro comparisons of the form, function, fertility, compatibility and competitiveness of farm versus wild Atlantic salmon sperm and eggs, in conditions mimicking the natural gametic microenvironment, using fish raised under similar environmental conditions. Despite selective domestication and reduced genetic diversity, we find functional equivalence in all farm fish gamete traits compared with their wild ancestral strain. Our results identify a clear threat of farm salmon reproduction with wild fish and therefore encourage further consideration of using triploid farm strains with optimized traits for aquaculture and fish welfare, as triploid fish remain reproductively sterile following escape. © 2014 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License (CC BY 3.0) (https://creativecommons.org/licenses/by/3.0/)

In the majority of insects, sperm fertilize the egg via a narrow canal through the outer chorion called the micropyle. Despite having this one primary function, there is considerable unexplained variation in the location, arrangement and number of micropyles within and between species. Here, we examined the relationship between micropyle number and female mating pattern through a comparative analysis across Lepidoptera. Three functional hypotheses could explain profound micropylar variation: (i) increasing micropyle number reduces the risk of infertility through sperm limitation in species that mate infrequently; (ii) decreasing micropyle number reduces the risk of pathological polyspermy in species that mate more frequently; and (iii) increasing micropyle number allows females to exert greater control over fertilization within the context of post-copulatory sexual selection, which will be more intense in promiscuous species. Micropyle number was positively related to the degree of female promiscuity as measured by spermatophore count, regardless of phylogenetic signal, supporting the hypothesis that micropyle number is shaped by post-copulatory sexual selection. We discuss this finding in the context of cryptic female choice, sperm limitation and physiological polyspermy.

Despite evidence that variation in male–female reproductive compatibility exists in many fertilization systems, identifying mechanisms of cryptic female choice at the gamete level has been a challenge. Here, under risks of genetic incompatibility through hybridization, we show how salmon and trout eggs promote fertilization by conspecific sperm. Using in vitro fertilization experiments that replicate the gametic microenvironment, we find complete interfertility between both species. However, if either species’ ova were presented with equivalent numbers of both sperm types, conspecific sperm gained fertilization precedence. Surprisingly, the species’ identity of the eggs did not explain this cryptic female choice, which instead was primarily controlled by conspecific ovarian fluid, a semiviscous, protein‐rich solution that bathes the eggs and is released at spawning. Video analyses revealed that ovarian fluid doubled sperm motile life span and straightened swimming trajectory, behaviors allowing chemoattraction up a concentration gradient. To confirm chemoattraction, cell migration tests through membranes containing pores that approximated to the egg micropyle showed that conspecific ovarian fluid attracted many more spermatozoa through the membrane, compared with heterospecific fluid or water. These combined findings together identify how cryptic female choice can evolve at the gamete level and promote reproductive isolation, mediated by a specific chemoattractive influence of ovarian fluid on sperm swimming behavior., This work was funded by the Natural Environment Research Council and the Royal Society. The authors declare no competing interests, financial or otherwise.

Theory predicts that males should evolve mechanisms to assess competition and allocate resources accordingly. This requires phenotypic plasticity, to accurately match responses to the environment. Plastic responses in males to sexual competition are diverse and widespread. However, our ability to understand and predict how they evolve is limited because their benefits are rarely measured, and costs are, as yet, entirely unquantified. In the fruit fly Drosophila melanogaster, males that anticipate strong competition for matings or fertilizations subsequently mate for longer and transfer more of two key seminal fluid proteins. This results in significantly elevated reproductive output. In this study, we examined the fitness effects of male responses to rivals across the entire male life span. Males were exposed to rivals or not throughout life while controlling mating opportunities. Males showed significant responses to rivals throughout their lifetimes, associated with significant early-life fitness benefits. However, these disappeared after the third mating. There were also significant costs—males exposed to rivals took significantly fewer mating opportunities in later life and had significantly shorter life spans than controls. The data suggest that there are substantial costs for males of mounting plastic responses to the threat of sexual competition.

Parasites impose strong selection on hosts to defend themselves, which is expected to result in trade-offs with other fitness traits such as reproduction. Here we test for genetic trade-offs between reproductive traits and immunity using Tribolium castaneum lines that were subject to experimental evolution. The lines have been exposed to contrasting sexual selection intensities via different sex ratios (female-biased, equal and male-biased). After 56 generations, the lines have significantly diverged and those experiencing high sexual selection have evolved males who are superior competitors for reproductive success, and females who are more resistant to multiple mating. All selected lines were assessed for both an immune measurement (phenoloxidase (PO) activity) and host resistance to the microsporidian Nosema whitei after two generations of relaxed selection. In contrast to our expectations we did not find any evidence for a genetic trade-off between investment in reproduction and immunity. Both PO and Nosema resistance did not differ between lines, despite their divergences in reproductive investment due to variation in sexual selection and conflict. Nevertheless, overall we found that females had higher PO activities and in the Nosema free control treatment survived longer than males, suggesting that females generally invest more in PO and survival under control conditions than males. This result fits the Bateman’s principle, which states that females gain fitness through increased immunity and longevity, while males gain fitness through increased mating success.

Behavioural plasticity allows animals to attune their behaviour to rapid environmental changes. Here we focus on plasticity in male mating behaviour in response to socio-sexual conditions. We discuss existing theory, generate predictions to facilitate exploration of the benefits of plastic behaviour, and identify parameters with the highest leverage on fitness. Existing data are synthesised to assess whether plasticity occurs pre- and post-copulation, to determine the direction of changes in behaviour, and to examine if plastic behaviour is fully flexible. We find that plasticity in males is widespread and not dominated by responses that occur pre- or post-copulation. Our synthesis also highlights areas that are underexplored, such as the limited data on the ultimate fitness consequences of such plastic behaviour.

SummaryAcross many species, males exhibit plastic responses when they encounter mating rivals [1]. The ability to tailor responses to the presence of rivals allows males to increase investment in reproduction only when necessary. This is important given that reproduction imposes costs [2] that limit male reproductive capacity, particularly when sperm competition occurs [3, 4]. Fruitfly (Drosophila melanogaster) males exposed to rivals subsequently mate for longer and thus accrue fitness benefits under increased competition [5], in line with theory [6, 7]. Here, we show that male D. melanogaster detect rivals by using a suite of cues and that the resulting responses lead directly to significant fitness benefits. We used multiple techniques to systematically remove auditory, olfactory, tactile, and visual cues, first singly and then in all possible combinations. No single cue alone was sufficient to allow males to detect rivals. However, the perception of any two cues from sound, smell, or touch permitted males to detect and respond adaptively to rivals through increased offspring production. Vision was only of marginal importance in this context. The findings indicate adaptive redundancy through the use of multiple, but interchangeable, cues. We reveal the robust mechanisms by which males assess their socio-sexual environment to precisely attune responses via the expression of plastic behavior.

Between-individual variance in potential reproductive rate theoretically creates a load in reproducing populations by driving sexual selection of male traits for winning competitions, and female traits for resisting the costs of multiple mating. Here, using replicated experimental evolution under divergent operational sex ratios (OSR, 9:1 or 1:6 ♀:♂) we empirically identified the parallel reproductive fitness consequences for females and males in the promiscuous flour beetle Tribolium castaneum. Our results revealed clear evidence that sexual conflict resides within the T. castaneum mating system. After 20 generations of selection, females from female-biased OSRs became vulnerable to multiple mating, and showed a steep decrease in reproductive fitness with an increasing number of control males. In contrast, females from male-biased OSRs showed no change in reproductive fitness, irrespective of male numbers. The divergence in reproductive output was not explained by variation in female mortality. Parallel assays revealed that males also responded to experimental evolution: individuals from male-biased OSRs obtained 27% greater reproductive success across 7-day competition for females with a control male rival, compared to males from the female-biased lines. Subsequent assays suggest that these differences were not due to postcopulatory sperm competitiveness, but to precopulatory/copulatory competitive male mating behavior.

Triploidy could prevent escaped farm salmon breeding in the wild, while also improving nutrient quality within farmed fillets. Despite these potential advantages, triploid Atlantic salmon have not been widely used in aquaculture, and their reproductive function has yet to be fully evaluated. Here, we compare reproductive function and fillet composition between triploid and diploid farm salmon under standard aquaculture rearing conditions. We show that female triploids are sterile and do not develop gonads. By contrast, males produce large numbers of motile spermatozoa capable of fertilizing wild salmon eggs. However, compared with diploids, reproductive development and survival rates of eggs fertilized by triploid males were significantly reduced, with less than 1% of eggs sired by triploid males reaching late-eyed stages of development. Analyses of fillets showed that total lipid and fatty acid quantities were significantly lower in triploid than in diploid Atlantic salmon fillets. However, when fatty acids were normalized to total lipid content, triploid fillets had significantly higher relative levels of important omega-3 longchain polyunsaturated fatty acids. Our results show that: (i) escaped triploid farm salmon are very unlikely to reproduce in the wild and (ii) if able to match diploid fillet lipid content, triploid farm salmon could achieve better fillet quality in terms of essential fatty acids. aquaculture, nutrition, polyploidy, sustainability, sterility, sperm

Invariant life-history theory has been used to identify parallels in life histories across diverse taxa. One important invariant life-history model predicts that, given simple assumptions and conditions, size-at-sex-change relative to maximum attainable body size (relative size-at-sex-change, RSSC) will be invariant across populations and species in sequential hermaphrodites. Even if there are broad species-wide limits to RSSC, populations could fine-tune RSSC to local conditions and, consequently, exhibit subtle but important differences in timing of sex change. Previous analyses of the invariant sex-change model have not explicitly considered the potential for meaningful differences in RSSC within the confines of a broader ‘invariance’. Furthermore, these tests differ in their geographical and taxonomic scope, which could account for their conflicting conclusions. We test the model using several populations of three female-first sex-changing Caribbean parrotfish species. We first test for species-wide invariance using traditional log–log regressions and randomisation analyses of population-specific point estimates of RSSC. We then consider error around these point estimates, which is rarely incorporated into invariant analyses, to test for differences among populations in RSSC. Log–log regressions could not unequivocally diagnose invariance in RSSC across populations; randomisation tests identified an invariant RSSC in redband parrotfish only. Analyses that incorporated within-population variability in RSSC revealed differences among populations in timing of sex change, which were independent of geography for all species. While RSSC may be evolutionarily constrained (as in redband parrotfish), within these bounds the timing of sex change may vary among populations. This variability is overlooked by traditional invariant analyses and not predicted by the existing invariant model.

Many populations of groupers (Teleostei: Serranidae) are overfished, partly because most species form spawning aggregations that are temporally and spatially predictable and therefore easily targeted by fisheries. However, most grouper fisheries operate year-round, thus there can also be high mortality during non-spawning periods. We investigated the impact of fishing around Anguilla, British West Indies, on a commercially important grouper, the red hind Epinephelus gutta- tus, during the non-breeding season. We combined information on the spatial intensity of the fishery with underwater surveys of groupers to test for associations between fishing intensity and fish size and density across 19 sites. Red hind density was unrelated to fishing intensity but red hinds were larger in areas that were targeted more intensively by fishers. While these results might be taken to suggest that fishing has no negative impacts on red hind demographics, we present evidence from fish markets that fishing intensity on this species during the non-spawning season is high. A variety of mechanisms may mask site-specific negative impacts on density and size of red hinds. In particu- lar, fishers can easily move among sites to track grouper abundance and body size, thereby making it difficult to detect impacts on red hinds during the non-spawning season.

Spermatozoal haplotypic DNA is prone to damage, leading to male fertility problems. So far, the assessment of sperm DNA breakage has been challenging because protamines render the nuclear chromatin highly compacted. Here, we report the application of a new test to quantify DNA fragmentation in spermatozoa of an externally fertilizing teleost fish. The sperm chromatin dispersion (SCD) test uses a species-specific lysing solution to generate controlled protein depletion that, followed by DNA-specific fluorescent labelling, allows an easy morphological discrimination between nuclei affected by DNA damage. Using tench (Tinca tinca) as our model, we first trialled the test against established, but more technically demanding, assays employingin situnick translation (ISNT) and the comet assay. The SCD test showed high concordance with ISNT, comet assay measures and a chromatin-swelling test, confirming the application of this straightforward SCD technique to various aspects of reproductive biology. Second, we examined between-male variation in DNA damage, and measured changes through time following spermatozoal activation. Between-male variation in the basal levels of average DNA damage ranged from 0 to 20% of sperm showing damage, and all showed increases in DNA fragmentation through time (0–60 min). The rates of DNA damage increase are the fastest so far recorded in sperm for a living organism, and may relate to the external fertilization mode. Our findings have relevance for broodstock selection and optimizing IVF protocols routinely used in modern aquaculture.

Polyandry and post-copulatory sexual selection provide opportunities for the evolution of female differential sperm selection. Here, we examined the influence of variation in major histocompatibility (MH) class I allelic composition upon sperm competition dynamics in Atlantic salmon. We ran in vitro fertilization competitions that mimicked the gametic microenvironment, and replicated a paired-male experimental design that allowed us to compare differences in sperm competition success among males when their sperm compete for eggs from females that were genetically either similar or dissimilar at the MH class I locus. Concurrently, we measured variation in spermatozoal traits that are known to influence relative fertilization success under these conditions. Contrary to the findings demonstrating mechanisms that promote MH complex heterozygosity, our results showed that males won significantly greater relative fertilization success when competing for eggs from genetically similar females at the MH class I. This result also showed covariation with the known influences of sperm velocity on relative fertilization success. We discuss these unexpected findings in relation to sperm–egg recognition and hybridization avoidance mechanisms based upon immunogenetic variation.

The polymorphic coral reef fish genus Hypoplectrus (hamlets) provides an excellent sys- tem for examining the initial stages of natural biological divergence in the expansive marine environ- ment. Despite close genetic similarities, hamlets occur in assortatively mating colour morphotypes. In this study, we determined whether ecological differences exist between morphs that could reinforce the assortative mating pattern within morphs. We compared the carbon and nitrogen stable isotope dietary signatures of 6 hamlet morphotypes from 5 geographically distant locations. Across 364 indi- viduals, and with the exception of fish sampled in the Gulf of Mexico, no significant isotopic associa- tions with morphotype were detected. Our results therefore provide no evidence that different hamlet morphs are associated with distinct dietary niches, despite finding highly significant geo- graphical differentiation for both isotopes. We argue that tight assortative mating without ecological divergence could be maintained through the demands of reciprocal cooperation within a reproduc- tive pattern of simultaneous hermaphroditism which characterises all hamlets.

Sex change is widespread among marine fishes, including many species that are fished heavily, and is thought to be of conservation concern under some circumstances. As such, an important question in conservation is whether the implementation of marine protected areas (MPAs), which is a commonly used marine conservation tool, works as effectively for sex-changers as for non-sex-changers. To address this issue, we used meta-analyses of the ratio of fish abundances inside vs. outside MPAs to determine whether sex change affects the extent to which fish densities respond to protection. When all data were considered, there were similar responses to protection irrespective of reproductive mode. However, when analyses were restricted to older reserves (at least 10 years’ protection), female-first sex-changers consistently benefited from protection. Non-sex-changers and male-first sex-changers showed more variable responses to protection and, as a result, there were no significant differences between fish with different reproductive modes in their overall response to protection. The same results were observed when the effects of fisheries status (targeted vs. not targeted) were controlled. Our results support the use of MPAs as important components of conservation and demonstrate that old reserves are most consistently beneficial to female-first sex-changing species. Finally, our results highlight the fact that some effects of protection are only detectable after several generations.

Assortative mate choice is, so far, the only demonstrated isolating barrier between colour morphs in fish species flocks, such as freshwater cichlids and marine hamlets, suggesting an important role for sexual selection in speciation. However, there has been little consideration of post-copulatory mechanisms that are known to influence reproductive isolation in other taxa. Selective pressures through a cost to hybrid matings, or genetic drift, may lead to the evolution of fertilization barriers through gamete incompatability. In cases of incipient speciation, such as the hamlets, complete fertilization blocks may not have evolved. Instead, differential fertilization between species may reduce the success of interspecific fertilizations. We examined the fertilization dynamics of sympatric Hypoplectrus nigricans (black hamlet) and Hypoplectrus puella (barred hamlet). Experimental crosses were performed to score fertilization success in within-morph and between-morph crosses. Fertilization success did not differ between self-fertilized, within-morph or between-morph crosses. We therefore found no evidence of post-mating barriers through sperm:egg incompatibilities and potential for self-fertilization. Our results are consistent with a mechanism of colour morph divergence based on sexually selected morph mating preferences.

Current theory to explain the adaptive significance of sex change over gonochorism predicts that female-first sex change could be adaptive when relative reproductive success increases at a faster rate with body size for males than for females. A faster rate of reproductive gain with body size can occur if larger males are more effective in controlling females and excluding competitors from fertilizations. The most simple consequence of this theoretical scenario, based on sexual allocation theory, is that natural breeding sex ratios are expected to be female biased in female-first sex changers, because average male fecundity will exceed that of females. A second prediction is that the intensity of sperm competition is expected to be lower in female-first sex-changing species because larger males should be able to more completely monopolize females and therefore reduce male-male competition during spawning. Relative testis size has been shown to be an indicator of the level of sperm competition, so we use this metric to examine evolutionary responses to selection from postcopulatory male-male competition. We used data from 116 comparable female-first sex-changing and nonhermaphroditic (gonochoristic) fish species to test these two predictions. In addition to cross-species analyses we also controlled for potential phylogenetic nonindependence by analyzing independent contrasts. As expected, breeding sex ratios were significantly more female biased in female-first sex-changing than nonhermaphroditic taxa. In addition, males in female-first sex changers had significantly smaller relative testis sizes that were one-fifth the size of those of nonhermaphroditic species, revealing a new evolutionary correlate of female-first sex change. These results, which are based on data from a wide range of taxa and across the same body-size range for either mode of reproduction, provide direct empirical support for current evolutionary theories regarding the benefits of female-first sex change.

Sex change is widespread among tropical marine fishes, many of which are targeted by fisheries. Conservation concerns have been raised that sex-changing species may be particularly prone to overexploitation by size-selective fishing. In the case of male-first sex-changers, populations may become egg limited if large females are disproportionately killed. However, if males reduce the size at which they change sex in response to higher female mortality, the population may still be sufficiently productive. We develop an age-based model to explore the effects of fishing on two types of male-first sex-changing fish: one with flexibility in size-atsex-change and one without. These effects were compared with those of non-sexchanging populations with similar life-history and population characteristics. The model predicts that if male-first sex-changers cannot respond to elevated female mortality by adjusting their size-at-sex-change, the population will be more prone to recruitment limitation and extinction than non-sex-changers. These effects will be amplified as smaller individuals become susceptible to fishing mortality. However, if size-at-sex-change is flexible, sex-changers may be as resilient to fishing as non-sex-changers. Knowledge of a species’ size-at-sex-change, and the mechanisms controlling it, should be fundamental to the selection of fisheries conservation strategies.

In vitro paired-male sperm competition experiments in Atlantic salmon Salmo salar for a single female’s eggs revealed that 2 s delays in sperm release caused significant reductions in paternity, with second males achieving only 30% fertilization success (against an expected 50%). This first-male fertilization precedence supports previous work suggesting that sperm competition follows the principles of a race in Atlantic salmon, and suggests that any timing asymmetry in sperm release within natural competitive spawnings could have significant consequences for male fertilization success.

Rensch's rule proposes a universal allometric scaling phenomenon across species where sexual size dimorphism (SSD) has evolved: in taxa with male-biased dimorphism, degree of SSD should increase with overall body size, and in taxa with female-biased dimorphism, degree of SSD should decrease with increasing average body size. Rensch's rule appears to hold widely across taxa where SSD is male-biased, but not consistently when SSD is female-biased. Furthermore, studies addressing this question within species are rare, so it remains unclear whether this rule applies at the intraspecific level. We assess body size and SSD within Tribolium castaneum (Herbst), a species where females are larger than males, using 21 populations derived from separate locations across the world, and maintained in isolated laboratory culture for at least 20 years. Body size, and hence SSD patterns, are highly susceptible to variations in temperature, diet quality and other environmental factors. Crucially, here we nullify interference of such confounds as all populations were maintained under identical conditions (similar densities, standard diet and exposed to identical temperature, relative humidity and photoperiod). We measured thirty beetles of each sex for all populations, and found body size variation across populations, and (as expected) female-biased SSD in all populations. We test whether Rensch's rule holds for our populations, but find isometry, i.e. no allometry for SSD. Our results thus show that Rensch's rule does not hold across populations within this species. Our intraspecific test matches previous interspecific studies showing that Rensch's rule fails in species with female-biased SSD.

Reproduction through sex carries substantial costs, mainly because only half of sexual adults produce offspring1. It has been theorized that these costs could be countered if sex allows sexual selection to clear the universal fitness constraint of mutation load2,3,4. Under sexual selection, competition between (usually) males and mate choice by (usually) females create important intraspecific filters for reproductive success, so that only a subset of males gains paternity. If reproductive success under sexual selection is dependent on individual condition, which is contingent to mutation load, then sexually selected filtering through ‘genic capture’5 could offset the costs of sex because it provides genetic benefits to populations. Here we test this theory experimentally by comparing whether populations with histories of strong versus weak sexual selection purge mutation load and resist extinction differently. After evolving replicate populations of the flour beetle Tribolium castaneum for 6 to 7 years under conditions that differed solely in the strengths of sexual selection, we revealed mutation load using inbreeding. Lineages from populations that had previously experienced strong sexual selection were resilient to extinction and maintained fitness under inbreeding, with some families continuing to survive after 20 generations of sib × sib mating. By contrast, lineages derived from populations that experienced weak or non-existent sexual selection showed rapid fitness declines under inbreeding, and all were extinct after generation 10. Multiple mutations across the genome with individually small effects can be difficult to clear, yet sum to a significant fitness load; our findings reveal that sexual selection reduces this load, improving population viability in the face of genetic stress., We thank the Natural Environment Research Council and the Leverhulme Trust for financial support, D. Edward for statistical advice and colleagues at the 2013 Biology of Sperm meeting for comments that improved analytical design and interpretation.

Summary 1Genes for insecticide resistance are usually traded against other fitness traits. However, in Tribolium castaneum, the opposite effect occurs under malathion-specific resistance: susceptible individuals show reduced reproductive success compared with resistant lines. 2To determine the mechanisms within this unusual relationship, we explored male fertilization success after sperm competition between homozygotes of two isogenic lines that were only either resistant or susceptible to malathion. We also measured key male reproductive traits known to influence sperm competition and reproductive success. 3Malathion-resistant males were superior sperm competitors than susceptible ones, which explains the rapid proliferation of the resistant genotype. This reproductive advantage was associated with increased ejaculate sperm number and sperm length for males carrying the resistance gene. Conversely, susceptible males have absolutely and relatively larger testis size. 4Resistance to malathion is conferred by an increase in activity of a qualitatively variant detoxifying carboxylesterase enzyme compared to the ‘original’ enzyme composition in susceptible populations. Carboxylesterases also play important and conserved roles in sperm development, maturation and possibly fertilization in different taxa. 5Our findings reveal a potential pleiotropic link between a single gene that codes for both insecticide resistance and influences key male reproductive traits through the regulation of carboxylesterase production.

In species where there is postcopulatory competition between males in the form of sperm competition, theory predicts that males should show prudent sperm allocation. Specifically, males should adjust the number of sperm that they inseminate in response to variations in the risk and intensity of sperm competition. Some experimental evidence for nonmammalian species indicates that ejaculate expenditure can be dynamic and adjusted in accordance with sperm competition theory. However, most studies have not used within-subject experimental designs to demonstrate ejaculate adjustments by individual males, and there have been no unequivocal demonstrations of prudent sperm allocation in mammals. In the present study, copulatory ejaculates were collected from 12 male rats that each mated under two experimental conditions: alone in a cage with a female (monandrous) and (accompanied by a ‘rival’ male who observed the copulation from behind a perforated plastic screen (polyandrous). As predicted by sperm competition theory, males ejaculated significantly more sperm when copulating in the presence of a rival than when copulating alone. Furthermore, the magnitudes of these ejaculate adjustments were highly predictable and were relatively consistent across individual males.

Sperm competition occurs when sperm from more than one male compete for fertilizations. This form of post-copulatory sexual selection is recognized as a significant and widespread force in the evolution of male reproductive biology and as a key determinant of differential male reproductive success. Despite its importance, however, detailed mechanisms of sperm competition at the gamete level remain poorly understood. Here, we use natural variation in spermatozoal traits among wild Atlantic salmon (Salmo salar), a species naturally adapted to sperm competition, to examine how the relative influences of sperm (i) number, (ii) velocity, (iii) longevity, and (iv) total length determine sperm competition success. Atlantic salmon fertilize externally, and we were therefore able to conduct controlled in vitro fertilization competitions while concurrently measuring spermatozoal traits within the aqueous micro-environment to which salmon gametes are naturally adapted. Microsatellite DNA fingerprinting revealed that a male's relative sperm velocity was the primary determinant of sperm competition success. There was no significant relationship between fertilization success and either relative sperm number or total length; sperm longevity showed an inverse relationship with competition success. These relationships were consistent for two experimental repeats of the in vitro fertilization competitions. Our results therefore show, under the natural microenvironment for salmon gametes, that relative sperm velocity is a key spermatozoal component for sperm competition success. Atlantic salmon sperm can be considered to enter a competition analogous to a race in which the fastest sperm have the highest probability of success.

Sperm competition, when sperm from different males compete to fertilize a female's ova [1], is a widespread and fundamental force in the evolution of animal reproduction [2]. The earliest prediction of sperm competition theory was that sperm competition selected for the evolution of numerous, tiny sperm, and that this force maintained anisogamy [3]. Here, we empirically test this prediction directly by using selective breeding to generate controlled and independent variance in sperm size and number traits in the cricket Gryllus bimaculatus. We find that sperm size and number are male specific and vary independently and significantly. We can therefore noninvasively screen individuals and then run sperm competition experiments between males that differ specifically in sperm size and number traits. Paternity success across 77 two-male sperm competitions (each running over 30-day oviposition periods) shows that males producing both relatively small sperm and relatively numerous sperm win competitions for fertilization. Decreased sperm size and increased sperm number both independently predicted sperm precedence. Our findings provide direct experimental support for the theory that sperm competition selects for maximal numbers of miniaturized sperm [3]. However, our study does not explain why G. bimaculatus sperm length persists naturally at ∼1 mm; we discuss possibilities for this sperm size maintenance.

Understanding why there is extensive variation in sperm form and function across taxa has been a challenge because sperm are specialized cells operating at a microscopic level in a complex environment. This comparative study collates published data to determine whether the evolution of sperm morphometry (sperm total length and separate component dimensions) is associated with sperm competition (when different males' sperm mix and compete for a female's ova) across 83 mammalian species. We use relative testes mass as an indicator of the intensity of sperm competition across taxa: relative investment into testes is widely accepted to predict the level of sperm competition that a species or population endures. Although we found evidence for positive associations between relative testes mass (controlling for allometry) and sperm morphometry across 83 mammalian species, these relationships were phylogenetically dependent. When we appropriately controlled for phylogenetic association using multiple regression within a phylogenetic framework, there was no relationship between relative testes mass and sperm length across mammals. Furthermore, we found no evidence for associations between relative testes mass and sperm head, mid-piece or flagellar lengths, nor was there a relationship with mid-piece or mitochondrial volumes. Results, therefore, indicate that sperm competition does not select for longer or shorter sperm across mammals, and alternative forces selecting on sperm form and function are discussed.