Potential Mating Partners Research Articles

Is promiscuity the key? Multiple paternity in the garden dormouse (Eliomys quercinus)

Many mammals have a promiscuous mating system with multiple sired litters. Promiscuity can increase the genetic variability, reduce the risk of inbreeding, and increase the effective population size, and is therefore crucial for preventing genetic loss and maintaining adaptability. This is particularly true for small and threatened populations. The garden dormouse (Eliomys quercinus) is a threatened species, which exhibited a drastic decline over the last 20–30 years. The aim of this study was therefore to investigate the mating system of the garden dormouse in mountain forest habitat by parentage analyses using 5 polymorphic microsatellite markers combined with morphometric data and information about the nesting behavior. Genetic parentage analyses revealed that 64% (9 of 14) of the litters were sired by at least two males, suggesting that garden dormice have a promiscuous mating system. The genetic findings were further supported by indirect indicators of promiscuity, such as testes, that were nearly four times larger than predicted for a rodent of its body mass and only slight male biased sexual size dimorphism. The finding of a promiscuous mating system in garden dormice should be taken into account in future conservation efforts. Due to its habitat preferences and limited dispersal potential garden dormice are vulnerable to forest fragmentation. The connection of suitable habitats facilitates dispersal and promotes access to potential mating partners, which could be especially important for populations colonizing new habitats. Access to potential mates may also reduce inbreeding, loss of genetic variability which is crucial for populations viability and survival.

Localized Expression of Olfactory Receptor Genes in the Olfactory Organ of Common Minke Whales.

Baleen whales (Mysticeti) possess the necessary anatomical structures and genetic elements for olfaction. Nevertheless, the olfactory receptor gene (OR) repertoire has undergone substantial degeneration in the cetacean lineage following the divergence of the Artiodactyla and Cetacea. The functionality of highly degenerated mysticete ORs within their olfactory epithelium remains unknown. In this study, we extracted total RNA from the nasal mucosae of common minke whales (Balaenoptera acutorostrata) to investigate ORs' localized expression. All three sections of the mucosae examined in the nasal chamber displayed comparable histological structure. However, the posterior portion of the frontoturbinal region exhibited notably high OR expression. Neither the olfactory bulb nor the external skin exhibited the expression of these genes. Although this species possesses four intact non-class-2 ORs, all the ORs expressed in the nasal mucosae belong to class-2, implying the loss of aversion to specific odorants. These anatomical and genomic analyses suggest that ORs are still responsible for olfaction within the nasal region of baleen whales, enabling them to detect desirable scents such as prey and potential mating partners.

Cip1, a CDK regulator, determines heterothallic mating or homothallic selfing in a protist

Mating type (sex) plays a crucial role in regulating sexual reproduction in most extant eukaryotes. One of the functions of mating types is ensuring self-incompatibility to some extent, thereby promoting genetic diversity. However, heterothallic mating is not always the best mating strategy. For example, in low-density populations or specific environments, such as parasitic ones, species may need to increase the ratio of potential mating partners. Consequently, many species allow homothallic selfing (i.e., self-fertility or intraclonal mating). Throughout the extensive evolutionary history of species, changes in environmental conditions have influenced mating strategies back and forth. However, the mechanisms through which mating-type recognition regulates sexual reproduction and the dynamics of mating strategy throughout evolution remain poorly understood. In this study, we show that the Cip1 protein is responsible for coupling sexual reproduction initiation to mating-type recognition in the protozoal eukaryote Tetrahymena thermophila. Deletion of the Cip1 protein leads to the loss of the selfing-avoidance function of mating-type recognition, resulting in selfing without mating-type recognition. Further experiments revealed that Cip1 is a regulatory subunit of the Cdk19-Cyc9 complex, which controls the initiation of sexual reproduction. These results reveal a mechanism that regulates the choice between mating and selfing. This mechanism also contributes to the debate about the ancestral state of sexual reproduction.

Sexual selection: competition for resources provided by mating partners

Abstract Ever since Darwin’s pioneering work, the definition of sexual selection has been subject to recurrent controversies. The main focus of a more recent debate centers on whether or not sexual selection encompasses intra-sexual competition for resources other than gametes. Specifically, it has been proposed to define sexual selection as competition for access to gametes and to consider competition for any other resources provided by mates as forms of natural selection. In this review, I elaborate on several drawbacks of this gamete-centered approach arguing that it (1) implies an artificial split of pre-copulatory competition for mates into two indistinguishable processes, (2) hinders the identification and quantification of sexual selection and, therefore, its distinction from other forms of natural selection, (3) gives rise to an overly male-biased perception on sexual selection, and (4) does not conform to Darwin’s original conception. In an attempt to provide a definition, which is explicit regarding the ultimate target of intra-sexual competition, I propose to define sexual selection as competition for access to reproductive resources provided by potential mating partners. I specify the main characteristics of this alternative definition in the context of previous ones and discuss potential limitations.

Decoding the genetic and chemical basis of sexual attractiveness in parasitic wasps.

Attracting and securing potential mating partners is of fundamental importance for reproduction. Therefore, signaling sexual attractiveness is expected to be tightly coordinated in communication systems synchronizing senders and receivers. Chemical signaling has permeated through all taxa of life as the earliest and most widespread form of communication and is particularly prevalent in insects. However, it has been notoriously difficult to decipher how exactly information related to sexual signaling is encoded in complex chemical profiles. Similarly, our knowledge of the genetic basis of sexual signaling is very limited and usually restricted to a few case studies with comparably simple pheromonal communication mechanisms. The present study jointly addresses these two knowledge gaps by characterizing two fatty acid synthase genes, that most likely evolved by tandem gene duplication, simultaneously impacting sexual attractiveness and complex chemical surface profiles in parasitic wasps. Gene knock-down in female wasps dramatically reduces their sexual attractiveness coinciding with a drastic decrease in male courtship and copulation behavior. Concordantly, we found a striking shift of methyl-branching patterns in the female surface pheromonal compounds, which we subsequently demonstrate to be the main cause for the greatly reduced male response. Intriguingly, this suggests a potential coding mechanism for sexual attractiveness mediated by specific methyl-branching patterns in CHC profiles. So far, the genetic underpinnings of methyl-branched CHCs are not well understood despite their high potential for encoding information. Our study sheds light on how biologically relevant information can be encoded in complex chemical profiles and on the genetic basis of sexual attractiveness.

A Caenorhabditis elegans Male Pheromone Feminizes Germline Gene Expression in Hermaphrodites and Imposes Life-History Costs.

Sex pheromones not only improve the reproductive success of the recipients, but also impose costs, such as a reduced life span. The underlying mechanisms largely remain to be elucidated. Here, we show that even a brief exposure to physiological amounts of the dominant Caenorhabditis elegans male pheromone, ascr#10, alters the expression of thousands of genes in hermaphrodites. The most dramatic effect on the transcriptome is the upregulation of genes expressed during oogenesis and the downregulation of genes associated with male gametogenesis. This result reveals a way in which social signals help to resolve the inherent conflict between spermatogenesis and oogenesis in a simultaneous hermaphrodite, presumably to optimally align reproductive function with the presence of potential mating partners. We also found that exposure to ascr#10 increased the risk of persistent intestinal infections in hermaphrodites due to pathological pharyngeal hypertrophy. Thus, our study reveals ways in which the male pheromone can not only have beneficial effects on the recipients' reproduction, but also cause harmful consequences that reduce life span.

Olfactory sex preferences in six Estrildid Finch species

Avian courtship behaviour is essential to attract potential mating partners. Courtship behaviours can involve displays of different sensory modes. Sex discrimination is a crucial step and in many bird species, sexes differ in acoustic and visual traits, allowing sex discrimination. It has been shown only recently that in some species of Estrildid Finches, chemical cues are involved in social communication. Here, we investigated whether olfaction also plays a role in sex discrimination in Estrildid Finches. Investigating olfactory sex preferences as an indicator behaviour in six different Estrildid Finch species, we aimed to understand whether sex- and/or species-specific differences in olfactory preferences exists and whether olfactory sex preferences correspond to species-specific differences in sex-specific acoustic and visual displays, e.g., singing, plumage dimorphisms and courtship dance. Olfactory sex preferences were tested in a Y-Maze test. We found differences in scent preferences among the different species of Estrildid Finches. We discussed the behavioural pattern with respect to other species-specific traits. And their potential implications in a broader mate choice context.

Genomic determination of breeding systems and trans-specific evolution of HD MAT genes in suilloid fungi.

Studying the signatures of evolution can help to understand genetic processes. Here, we demonstrate how the existence of balancing selection can be used to identify the breeding systems of fungi from genomic data. The breeding systems of fungi are controlled by self-incompatibility loci that determine mating types between potential mating partners, resulting in strong balancing selection at the loci. Within the fungal phylum Basidiomycota, two such self-incompatibility loci, namely HD MAT locus and P/R MAT locus, control mating types of gametes. Loss of function at one or both MAT loci results in different breeding systems and relaxes the MAT locus from balancing selection. By investigating the signatures of balancing selection at MAT loci, one can infer a species' breeding system without culture-based studies. Nevertheless, the extreme sequence divergence among MAT alleles imposes challenges for retrieving full variants from both alleles when using the conventional read-mapping method. Therefore, we employed a combination of read-mapping and local de novo assembly to construct haplotypes of HD MAT alleles from genomes in suilloid fungi (genera Suillus and Rhizopogon). Genealogy and pairwise divergence of HD MAT alleles showed that the origins of mating types predate the split between these two closely related genera. High sequence divergence, trans-specific polymorphism, and the deeply diverging genealogy confirm the long-term functionality and multiallelic status of HD MAT locus in suilloid fungi. This work highlights a genomics approach to studying breeding systems regardless of the culturability of organisms based on the interplay between evolution and genetics.

Selection index theory for populations under directional and stabilizing selection

BackgroundThe purpose of a selection index is that its use to select animals for breeding maximizes the profit of a breed in future generations. The profit of a breed is in general a quantity that predicts the satisfaction of future owners with their breed, and the satisfaction of the consumers with the products that are produced by the breed. Many traits, such as conformation traits and product quality traits have intermediate optima. Traditional selection index theory applies only to directional selection and cannot achieve any further improvement once the trait means have reached their optima. A well-founded theory is needed that extends the established selection index theory to cover directional as well as stabilizing selection as limiting cases, and that can be applied to maximize the profit of a breed in both situations.ResultsThe optimum selection index shifts the trait means towards the optima and, in the case of stabilizing selection, decreases the phenotypic variance, which causes the phenotypes to be closer to the optimum. The optimum index depends not only on the breeding values, but also on the squared breeding values, the allele contents of major quantitative trait loci (QTL), the QTL heterozygosities, the inbreeding coefficient of the animal, and the kinship of the animal with the population.ConclusionThe optimum selection index drives the alleles of major QTL to fixation when the trait mean approaches the optimum because decreasing the phenotypic variance shifts the trait values closer to the optimum, which increases the profit of the breed. The index weight on the kinship coefficient balances the increased genetic gain that can be achieved in future generations by outcrossing, and the increased genetic gain that can be achieved under stabilizing selection by reducing the phenotypic variance. In a model with dominance variance, it can also account for the effect of inbreeding depression. The combining ability between potential mating partners, which predicts the total merit of their offspring, could become an important parameter for mate allocation that could be used to further shift the phenotypes towards their optimum values.

Male sex pheromone in the parasitoid wasp Nasonia longicornis: Chemical and behavioral analyses

The use of sex pheromones for the attraction of potential mating partners is widespread in insects. Species-specificity of these chemical signals is essential, particularly in closely related species with overlapping habitats. In parasitoid wasps of the genus Nasonia, it is the males that produce sex pheromones in their rectal vesicles. The genus consists of four species: N. vitripennis (Nv), N. giraulti (Ng), N. oneida (No), and N. longicornis (Nl). The cosmopolitan species Nv is sympatric with Ng and No in eastern North America and with Nl in the west. Interspecific courtship is common in Nasonia although hybridization is prevented in most combinations by Wolbachia-mediated cytoplasmic incompatibility. The pheromone of Nv males differs from all Nasonia congeners by an additional component that is believed to ensure precopulatory isolation from the sympatric congeners. Detailed investigations on these interactions, however, exist only for the species combination Nv/Ng. Here, we report the results of chemical and behavioral investigations on the sex pheromone of Nl males. The pheromone consists of (4R,5S)-5-hydroxy-4-decanolide (RS) and 4-methylquinazoline (MQ) as a minor component which are produced only after eclosion. Pheromone titers peaked 2 days after eclosion and remained constant on day three. The pheromone is deposited by abdomen dipping which males increasingly exhibited after mating or brief contact with a female. The presence of hosts containing females about to emerge did not increase marking behavior. Site fidelity of males at their own pheromone markings is mediated by MQ. Both natural and synthetic pheromone attracted virgin but not mated females and both RS and MQ are required for female attraction. Females collected during emergence from the host responded likewise to the pheromone and 84% of them produced mixed-sex offspring showing that most of them emerge unmated. Nl females preferred pheromone extract from conspecific males to extract from Nv males, and the addition of the Nv-specific component (4R,5R)-5-hydroxy-4-decanolide (RR) to the Nl pheromone made them unresponsive. The present study demonstrates that Nl uses the male sex pheromone in a similar way as previously shown for Nv and Ng. Furthermore, it shows that Nl females use the Nv-specific pheromone component RR to avoid costly sexual interactions with Nv males in regions of sympatry.

The serotonin circuit that coordinates germline proliferation and egg laying with other reproductive functions in Caenorhabditis elegans.

Behaviour and physiology are altered in reproducing animals, but neuronal circuits that regulate these changes remain largely unknown. Insights into mechanisms that regulate and possibly coordinate reproduction-related traits could be gleaned from the study of sex pheromones that can improve the reproductive success of potential mating partners. In Caenorhabditis elegans, the prominent male pheromone, ascr#10, modifies reproductive behaviour and several aspects of reproductive physiology in hermaphrodite recipients, including improving oocyte quality. Here we show that a circuit that contains serotonin-producing and serotonin-uptaking neurons plays a key role in mediating effects of ascr#10 on germline development and egg laying behaviour. We also demonstrate that increased serotonin signalling promotes proliferation of germline progenitors in adult hermaphrodites. Our results establish a role for serotonin in maintaining germline quality and highlight a simple neuronal circuit that acts as a linchpin that couples food intake, mating behaviour, reproductive output, and germline renewal and provisioning.

Mate choice is affected by parasite infestation rate of the choosing individual as well as of potential mating partners.

Parasites are known to be a key driving force in mate choice and are important for the expression and evolution of ornaments and behavioral traits being used. However, there is little experimental evidence on how the parasite's burden of the choosing individual is integrated into the mate-choice process and how it affects decision-making, especially in relation to parasite infestation of potential mates. Thus, the aim of our study was to determine whether female house sparrows Passer domesticus adjust their mate preference according to their own as well as the parasite load of prospective partners. To do this, we experimentally manipulated female parasite load and determined their mate preferences prior to and after parasite treatment. We manipulated the chronic coccidian parasite burden of females either by initiating the acute infection phase via re-infecting them with coccidian or by temporally reducing the parasite load of coccidia. We then measured the effect of this manipulation on mate preference by presenting females with a choice of four stimuli: three males with similar ornaments, but unmanipulated, naturally varying chronic coccidiosis levels, and an unmanipulated control female. Additionally, we recorded some males' behavior in relation to their infection status pointing toward an increased or reduced interest in mating. We found that females preferred highly infested males prior to manipulation, regardless of their own infestation level. However, after manipulation, infested females avoided highly infested males probably in response to the deterioration of their health condition by parasites. Our study suggests that mate-choice decisions are more complex when they are mediated by parasites. The implications of parasites for evolutionary theories of sexual signaling and mate choice are discussed.

Hearing, touching, and multisensory integration during mate choice.

Mate choice is a potent generator of diversity and a fundamental pillar for sexual selection and evolution. Mate choice is a multistage affair, where complex sensory information and elaborate actions are used to identify, scrutinize, and evaluate potential mating partners. While widely accepted that communication during mate assessment relies on multimodal cues, most studies investigating the mechanisms controlling this fundamental behavior have restricted their focus to the dominant sensory modality used by the species under examination, such as vision in humans and smell in rodents. However, despite their undeniable importance for the initial recognition, attraction, and approach towards a potential mate, other modalities gain relevance as the interaction progresses, amongst which are touch and audition. In this review, we will: (1) focus on recent findings of how touch and audition can contribute to the evaluation and choice of mating partners, and (2) outline our current knowledge regarding the neuronal circuits processing touch and audition (amongst others) in the context of mate choice and ask (3) how these neural circuits are connected to areas that have been studied in the light of multisensory integration.

Male mating preference in an ixodid tick

BackgroundMate choice is a fundamental element of sexual selection and has the potential to shape the evolution of traits. Mate choice based on body size has been shown to be a common trait in several arthropod species. In hard ticks, a taxon of medical and veterinary importance, engorgement weight is positively correlated with reproductive output but it is unknown whether adult males show mate choice. Here, we experimentally investigated whether males (i) use chemical cues to choose their mating partner, (ii) consistently choose for the same female individual and (iii) prefer females with highest weight after feeding.MethodsWe used two experimental setups which allowed chemical communication between ticks: (i) a horizontal tube preventing physical contact with the female and (ii) an arena where tactile cues were allowed. In total, we tested 62 different triads in 124 tests (66 tests in the horizontal tube and 58 in the arena) composed of one male that could choose between two engorged females. Specifically, we tested 42 triads in the tube and 46 in the arena; 24 triads were repeatedly tested in the tube while 38 triads were tested in both setups.ResultsWe found no preference for individual or heavier females in either setup. However, in the horizontal tube setup, males significantly preferred females that were not visited by them in the previous test.ConclusionsOur results suggest a lack of male mate choice despite heavier females having higher fecundity. However, future studies should take into account that males may recognize the potential mating partners they previously met.Graphical

A male pheromone that improves the quality of the oogenic germline

Pheromones exchanged by conspecifics are a major class of chemical signals that can alter behavior, physiology, and development. In particular, males and females communicate with potential mating partners via sex pheromones to promote reproductive success. Physiological and developmental mechanisms by which pheromones facilitate progeny production remain largely enigmatic. Here, we describe how a Caenorhabditis elegans male pheromone, ascr#10, improves the oogenic germline. Before most signs of aging become evident, C. elegans hermaphrodites start producing lower-quality gametes characterized by abnormal morphology, increased rates of chromosomal nondisjunction, and higher penetrance of deleterious alleles. We show that exposure to the male pheromone substantially ameliorates these defects and reduces embryonic lethality. ascr#10 stimulates proliferation of germline precursor cells in adult hermaphrodites. Coupled to the greater precursor supply is increased physiological germline cell death, which is required to improve oocyte quality in older mothers. The hermaphrodite germline is sensitive to the pheromone only during a time window, comparable in duration to a larval stage, in early adulthood. During this period, prereproductive adults assess the suitability of the environment for reproduction. Our results identify developmental events that occur in the oogenic germline in response to a male pheromone. They also suggest that the opposite effects of the pheromone on gamete quality and maternal longevity arise from competition over resource allocation between soma and the germline.

Pheromone Guidance of Polarity Site Movement in Yeast.

Cells’ ability to track chemical gradients is integral to many biological phenomena, including fertilization, development, accessing nutrients, and combating infection. Mating of the yeast Saccharomyces cerevisiae provides a tractable model to understand how cells interpret the spatial information in chemical gradients. Mating yeast of the two different mating types secrete distinct peptide pheromones, called a-factor and α-factor, to communicate with potential partners. Spatial gradients of pheromones are decoded to guide mobile polarity sites so that polarity sites in mating partners align towards each other, as a prerequisite for cell-cell fusion and zygote formation. In ascomycetes including S. cerevisiae, one pheromone is prenylated (a-factor) while the other is not (α-factor). The difference in physical properties between the pheromones, combined with associated differences in mechanisms of secretion and extracellular pheromone metabolism, suggested that the pheromones might differ in the spatial information that they convey to potential mating partners. However, as mating appears to be isogamous in this species, it is not clear why any such signaling difference would be advantageous. Here we report assays that directly track movement of the polarity site in each partner as a way to understand the spatial information conveyed by each pheromone. Our findings suggest that both pheromones convey very similar information. We speculate that the different pheromones were advantageous in ancestral species with asymmetric mating systems and may represent an evolutionary vestige in yeasts that mate isogamously.

Sexual inactivation induced by the mucus that covers land snail love darts: sexual selection and evolution of allohormones in hermaphrodites.

The transfer of male accessory gland secretions is a well-investigated reproductive strategy for winning in sexual selection. An example of such a strategy is the conspicuous mating behaviour of simultaneously hermaphroditic land snails, the so-called shooting of love darts, whereby a snail drives a love dart(s) into the body of its mating partner. In the land snail Euhadra quaesita, it has been shown that a specific mucus which coats the love dart is transferred into the partner's haemolymph and that it suppresses subsequent matings in the darted individual. However, how the mucus of the love dart suppresses rematings remains unclear. In the present study, we tested the hypothesis that by injection of the dart mucus, love-dart shooters manipulate the physiology of a dart recipient and make the individual sexually inactive. In an experiment in which snails were provided with opportunities to encounter a potential mating partner, we found that the latency period to achieve sexual arousal was longer in snails injected with the dart mucus than in snails of the control treatments. This finding indicates that the dart mucus delays sexual arousal in injected snails. This delay in arousal is a novel example of the effects of the mucus in simultaneously hermaphroditic land snails. The remating suppression effect of the dart mucus is likely to occur through sexual inactivation.

Facial threat affects trust more strongly than facial attractiveness in women than it does in men

Trust is essential in initiating social relationships. Due to the differential evolution of sex hormones as well as the fitness burdens of producing offspring, evaluations of a potential mating partner’s trustworthiness likely differ across sexes. Here, we explore unknown sex-specific effects of facial attractiveness and threat on trusting other-sex individuals. Ninety-three participants (singles; 46 women) attracted by the other sex performed an incentivized trust game. They had to decide whether to trust individuals of the other sex represented by a priori-created face stimuli gradually varying in the intensities of both attractiveness and threat. Male and female participants trusted attractive and unthreatening-looking individuals more often. However, whereas male participants’ trust behavior was affected equally by attractiveness and threat, female participants’ trust behavior was more strongly affected by threat than by attractiveness. This indicates that a partner’s high facial attractiveness might compensate for high facial threat in male but not female participants. Our findings suggest that men and women prioritize attractiveness and threat differentially, with women paying relatively more attention to threat cues inversely signaling parental investment than to attractiveness cues signaling reproductive fitness. This difference might be attributable to an evolutionary, biologically sex-specific decision regarding parental investment and reproduction behavior.

Does sexual experience affect the strength of male mate choice for high-quality females in Drosophila melanogaster?

Although females are traditionally thought of as the choosy sex, there is increasing evidence in many species that males will preferentially court or mate with certain females over others when given a choice. In the fruit fly, Drosophila melanogaster, males discriminate between potential mating partners based on a number of female traits, including species, mating history, age, and condition. Interestingly, many of these male preferences are affected by the male's previous sexual experiences, such that males increase courtship toward types of females that they have previously mated with and decrease courtship toward types of females that have previously rejected them. D. melanogaster males also show courtship and mating preferences for larger females over smaller females, likely because larger females have higher fecundity. It is unknown, however, whether this preference shows behavioral plasticity based on the male's sexual history as we see for other male preferences. Here, we manipulate the sexual experience of D. melanogaster males and test whether this manipulation has any effect on the strength of male mate choice for large females. We find that sexually inexperienced males have a robust courtship preference for large females that is unaffected by previous experience mating with, or being rejected by, females of differing sizes. Given that female body size is one of the most common targets of male mate choice across insect species, our experiments with D. melanogaster may provide insight into how these preferences develop and evolve.

Electrophysiological Recordings from the Olfactory Epithelium and Human Brain in Response to Stimulation with HLA Related Peptides

In many species, social communication and mate choice are influenced by olfactory cues associated with the major histocompatibility complex (MHC). It has been reported that humans also respond to olfactory signals related to the human MHC-equivalent, the Human Leucocyte Antigen (HLA)-System, and exhibit an olfactory-mediated preference for potential mating partners with a dissimilar, disassortative, HLA-type compared to their own. The aim of this study was to investigate whether HLA-associated peptides, presented as volatile cues, elicit neuronal responses at the receptors in the human olfactory epithelium and can be consciously perceived. To this end the discrimination ability for peptides was tested in a 3-alternative forced choice model. Furthermore electro-olfactograms of the olfactory epithelium and EEG-derived chemosensory event related potentials were recorded using precisely controlled olfactometric stimulation with peptides and control odors. Based on responses from 52 young, healthy participants the peptides could not be discriminated and the electrophysiological signals provided no evidence for a specific response to the peptides which was in contrast to the control odors. In conclusion, within the current setup the results suggest that HLA-associated peptides do not produce specific olfactory activation in humans.