Mechanisms Of Sperm Competition Research Articles

Using Gamma Irradiation to Predict Sperm Competition Mechanism in Bagrada hilaris (Burmeister) (Hemiptera: Pentatomidae): Insights for a Future Management Strategy.

The stink bug, Bagrada hilaris, is a pest of mainly Brassicaceae crops. It is native to Africa and Asia and was recently reported as invasive in the southwestern part of the USA and in South America. There are no mitigation programs in place that do not involve pesticides. Therefore, much attention has recently been paid to the study of this species in order to identify sustainable and effective control strategies, such as the Sterile Insect Technique (SIT). In order to evaluate the suitability of the SIT on this pest, the mechanism of post-copulatory sperm competition was investigated. This is a polyandrous species, and it is thus important to understand whether irradiated males are able to compete with wild, e.g., non-irradiated, males for sperm competition after matings. Sperm competition was studied by sequentially mating a healthy virgin female first with a non-irradiated male, and then with a γ-irradiated (Co-60) one, and again in the opposite order. Males were irradiated at three different doses: 60, 80, and 100 Gy. The fecundity and fertility of the females, in the two orders of mating, were scored in order to perform an initial assessment of the success of sperm competition with a P2 index. Sperm from the non-irradiated male were utilized at the lowest irradiation doses (60 and 80 Gy), whereas the irradiated sperm were preferentially utilized at the highest dose (100 Gy). Bagrada hilaris exhibited high variability in P2 indexes, indicating a sperm-mixing mechanism.

Fertilization mode drives sperm length evolution across the animal tree of life.

Evolutionary biologists have endeavoured to explain the extraordinary diversity of sperm morphology across animals for more than a century. One hypothesis to explain sperm diversity is that sperm length is shaped by the environment where fertilization takes place (that is, fertilization mode). Evolutionary transitions in fertilization modes may transform how selection acts on sperm length, probably by affecting postcopulatory mechanisms of sperm competition and the scope for cryptic female choice. Here, we address this hypothesis by generating a macro-evolutionary view of how fertilization mode (including external fertilizers, internal fertilizers and spermcasters) influences sperm length diversification among 3,233 species from 21 animal phyla. We show that sperm are shorter in species whose sperm are diluted in aquatic environments (that is, external fertilizers and spermcasters) and longer in species where sperm are directly transferred to females (that is, internal fertilizers). We also show that sperm length evolves faster and with a greater number of adaptive shifts in species where sperm operate within females (for example, spermcasters and internal fertilizers). Our results demonstrate that fertilization mode is a key driver in the evolution of sperm length across animals, and we argue that a complex combination of postcopulatory forces has shaped sperm length diversification throughout animal evolution.

Sperm competition in golden cuttlefish Sepia esculenta: The impact of mating order and male size

Sperm competition, as the last form of male competition prior to fertilization, is an important component in the reproductive and evolutionary biology of polyandrous species. Golden cuttlefish (Sepia esculenta) is a commercially important annual cephalopod, in which females often mate with multiple males during their mating season and maintain viable sperm in their sperm storage organs for a long time. This multiple-mating system leads to male competition and ultimately sperm competition. To explore the mechanisms of sperm competition in cuttlefish, a laboratory study was conducted using three experimental approaches. First, two similar-sized males were introduced to sequentially mate with a single female (experiment 1), to investigate mating order effects on paternity. Thereafter, a four-male experiment was run (experiment 2), which again tested for mating sequence on paternal contribution. Finally, to test whether male size is a factor affecting paternity, we compared the paternity of two different-sized males that were sequentially mated with a single female (experiment 3). The paternity of the offspring was analyzed using six microsatellite DNA markers on fertilized eggs. The results revealed last-male sperm precedence in all experiments, and no effect of male size was detected. For the two-male experiment, the last male to mate had a progeny share (P2) of 42.86%–61.90%. For the four-male experiment, the last mated male had the highest progeny share (P4) with a proportion of 35.56%–43.75%. Even with differential male sizes in the two-male experiment, the last mated male retained the highest progeny share (P2, 58.33%–91.67%). This study shows a consistent pattern of last-sperm advantage across different experiments and contributes to growing research on cephalopod mating systems. Proper mating schemes should be considered during seedling production for S. esculenta to ascertain efficient resource use and genetic diversity in produced offspring.

Mating failure shapes the patterns of sperm precedence in an insect

Estimates of last male sperm precedence (P2) are often used to infer mechanisms of sperm competition, a form of post-copulatory sexual selection. However, high levels of mating failure (i.e. copulations resulting in no offspring) in a population can lead to misinterpretations of sperm competition mechanisms. Through simulations, García-González (2004) illustrated how mating failure could cause bimodal distributions of paternity with peaks at P2 = 0 and 1, under a random sperm mixing mechanism. Here, we demonstrate this effect empirically with the seed bug Lygaeus simulans, a species known to exhibit high levels of mating failure (40–60%), using a morphological marker to estimate paternity. Contrary to previous findings in a sister species, we did not find strong evidence for last male sperm precedence. There was a tendency towards last male precedence (P2 = 0.58) but within the expected range for random sperm mixing. Instead, P2 was highly variable, with a bimodal distribution, as predicted by García-González (2004). After taking mating failure into account, the strongest driver of paternity outcome was copulation duration. Furthermore, we found evidence that mating failure could partly be a female-associated trait. Some doubly-mated females were more likely to produce no offspring or produce offspring from two different sires than expected by chance. Therefore, some females are more prone to experience mating failure than others, a result that mirrors an earlier result in male L. simulans. Our results confirm that mating failure needs to be considered when interrogating mechanisms of post-copulatory sexual selection.Significance statementMating failure arises when animals fail to produce offspring across their lifetime. This may be due to a failure to find a mate or a failure to produce offspring after one or more apparently successful matings. Sperm competition is when ejaculates of rival males compete to fertilize a female’s eggs. Estimates of second male paternity (P2) are often used to infer mechanisms of sperm competition (i.e. which male “wins” and how). However, García-González (2004) suggested that high levels of mating failure can skew paternity (i.e. give spuriously high/low levels of P2) and lead to misinterpretations of these mechanisms. We carried out sperm competition experiments on Lygaeus simulans seed bugs using a morphological marker to estimate paternity. We show empirically that mating failure does skew patterns of paternity, causing a bimodal distribution of P2. Therefore, by disrupting patterns of sperm competition, mating failure influences both the action of post-copulatory sexual selection and also our understanding of the mechanisms of sperm competition.

Genetic Factors Influencing Sperm Competition.

Females of many different species often mate with multiple males, creating opportunities for competition among their sperm. Although originally unappreciated, sperm competition is now considered a central form of post-copulatory male–male competition that biases fertilization. Assays of differences in sperm competitive ability between males, and interactions between females and males, have made it possible to infer some of the main mechanisms of sperm competition. Nevertheless, classical genetic approaches have encountered difficulties in identifying loci influencing sperm competitiveness while functional and comparative genomic methodologies, as well as genetic variant association studies, have uncovered some interesting candidate genes. We highlight how the systematic implementation of approaches that incorporate gene perturbation assays in experimental competitive settings, together with the monitoring of progeny output or sperm features and behavior, has allowed the identification of genes unambiguously linked to sperm competitiveness. The emerging portrait from 45 genes (33 from fruit flies, 8 from rodents, 2 from nematodes, and 2 from ants) is their remarkable breadth of biological roles exerted through males and females, the non-preponderance of sperm genes, and their overall pleiotropic nature.

A review of studies on the fire millipede genus centrobolus (diplopoda: trigoniulidae)

Studies on Centrobolus spp. were reviewed in which mechanisms of selection, sperm competition and cryptic female choice were studied. Approaches (1) quantify size dimorphism and find the selection pressures operating on the sexes, (2) determine the functional significance of male and female genitalia, (3) understand why there should be a conflict of sexual interests in prolonged copulations, and (4) resolve the mechanisms of sperm competition and cryptic female choice in comparing male mating strategies to female sperm usage were included.

LOSP: A putative marker of parasperm lineage in male reproductive system of the prosobranch mollusk Littorina obtusata.

Reproductive isolation is the key attribute of biological species and establishment of the reproductive barriers is an essential event for speciation. Among the mechanisms of reproductive isolation, gamete incompatibility due to the variability of gamete interaction proteins may drive fast divergence even in sympatry. However, the number of available models to study this phenomenon is limited. In case of internally fertilized invertebrates, models to study gamete incompatibility and sperm competition mechanisms are restricted to a single taxon: insects. Here, we propose a group of closely related Littorina species as a new model for such studies. Particularly since periwinkles are already thoroughly studied in terms of morphology, physiology, ecology, phylogeny, and ecological speciation. Earlier, we have identified the first species-specific Littorina sperm protein (LOSP) with no known conservative domains or homologies. LOSP is relatively abundant component of sperm extracts and might be involved in gamete incompatibility. Here, we characterize its definitive localization and mRNA expression pattern in the male reproductive system by immunocytochemistry and RNA in situ hybridization. We demonstrate that LOSP distribution is limited to the parasperm cells. Losp gene expression occurs only at the early stages of parasperm development. The protein is stored within granules of mature parasperm and, most likely, is released after ejaculation inside female reproductive system. Thus, LOSP is the only described molluscan paraspermal protein to date, and there is a possibility for LOSP to be involved in gamete incompatibility since heterospermy is a common phenomenon among Littorina.

X exceptionalism in Caenorhabditis speciation.

Speciation genetics research in diverse organisms shows the X-chromosome to be exceptional in how it contributes to "rules" of speciation. Until recently, however, the nematode phylum has been nearly silent on this issue, despite the model organism Caenorhabditis elegans having touched most other topics in biology. Studies of speciation with Caenorhabditis accelerated with the recent discovery of species pairs showing partial interfertility. The resulting genetic analyses of reproductive isolation in nematodes demonstrate key roles for the X-chromosome in hybrid male sterility and inviability, opening up new understanding of the genetic causes of Haldane's rule, Darwin's corollary to Haldane's rule, and enabling tests of the large-X effect hypothesis. Studies to date implicate improper chromatin regulation of the X-chromosome by small RNA pathways as integral to hybrid male dysfunction. Sexual transitions in reproductive mode to self-fertilizing hermaphroditism inject distinctive molecular evolutionary features into the speciation process for some species. Caenorhabditis also provides unique opportunities for analysis in a system with XO sex determination that lacks a Y-chromosome, sex chromosome-dependent sperm competition differences and mechanisms of gametic isolation, exceptional accessibility to the development process and rapid experimental evolution. As genetic analysis of reproductive isolation matures with investigation of multiple pairs of Caenorhabditis species and new species discovery, nematodes will provide a powerful complement to more established study organisms for deciphering the genetic basis of and rules to speciation.

Sperm removal during copulation confirmed in the oldest extant damselfly, Hemiphlebia mirabilis.

Postcopulatory sexual selection may favour mechanisms to reduce sperm competition, like physical sperm removal by males. To investigate the origin of sperm removal, I studied the reproductive behaviour and mechanisms of sperm competition in the only living member of the oldest damselfly family, Hemiphlebia mirabilis, one species that was considered extinct in the 1980s. This species displays scramble competition behaviour. Males search for females with short flights and both sexes exhibit a conspicuous “abdominal flicking”. This behaviour is used by males during an elaborate precopulatory courtship, unique among Odonata. Females use a similar display to reject male attempts to form tandem, but eventually signal receptivity by a particular body position. Males immobilise females during courtship using their legs, which, contrarily to other damselflies, never autotomise. Copulation is short (range 4.1–18.7 min), and occurs in two sequential stages. In the first stage, males remove part of the stored sperm, and inseminate during the second stage, at the end of mating. The male genital ligula matches the size and form of female genitalia, and ends by two horns covered by back-oriented spines. The volume of sperm in females before copulation was 2.7 times larger than the volume stored in females whose copulation was interrupted at the end of stage I, indicative of a significant sperm removal. These results point out that sperm removal is an old character in the evolution of odonates, possibly dating back to the Permian.

Gonopod mechanics in <em>Centrobolus</em> Cook (Spirobolida: Trigoniulidae) II. Images <em></em>

Gonopod mechanics were described for four species of millipedes in the genus Centrobolus and are now figured using scanning electron microscopy (SEM) with the aim to show the mechanism of sperm competition. Structures of sperm displacement include projections on a moveable telopodite and tips on a distal process (opisthomerite). Three significant contact zones between the male and female genitalia were recognized: (1) distal telopodite of the coleopod and the vulva, (2) phallopod and the bursa, (3) sternite and legs of the female.

Gonopod mechanics in Centrobolus Cook (Spirobolida: Trigoniulidae) II. Images

Gonopod mechanics were described for four species of millipedes in the genus Centrobolus and are now figured using scanning electron microscopy (SEM) with the aim to show the mechanism of sperm competition. Structures of sperm displacement include projections on a moveable telopodite and tips on a distal process (opisthomerite). Three significant contact zones between the male and female genitalia were recognized: (1) distal telopodite of the coleopod and the vulva, (2) phallopod and the bursa, (3) sternite and legs of the female.

Sperm production characteristics vary with level of sperm competition inCataglyphisdesert ants

SummaryUnder polyandry, males are selected to produce more competitive ejaculates. Theoretical models have explored how the mechanism of sperm competition drives males to partition investment within an ejaculate between sperm quantity and quality. The raffle‐based competition model predicts that increased level of sperm competition selects for larger numbers of sperm in ejaculates. Sperm competition is also thought to promote the evolution of longer sperm, because longer sperm could be faster.In eusocial Hymenoptera, the mating system imposes unique selective pressures on male ejaculates. Males are short lived; they reach adulthood with a finite amount of spermatozoa, and they mate typically with a single or a few females and die. The actual number of spermatozoa stored in their accessory testes at emergence is thus a reliable measure of total investment into sperm production. In a comparative study of 15 species ofCataglyphisdesert ants, we used phylogenetically controlled analyses to investigate relationships between levels of sperm competition, sperm production and sperm length. We measured sperm production by quantifying the number of spermatozoa present in testes, instead of using a proxy measure such as size of testes. Multiple queen mating is the ancestral state in the genus but reduction in mating frequency evolved secondarily and independently in some clades, providing a unique opportunity to examine how reduction from multiple to single mating influences sperm traits.Our results provide phylogenetically robust evidence that species experiencing greater levels of sperm competition produce more sperm. After controlling for male size, investment in sperm production decreases significantly according to the sequence obligatory multiple queen mating > multiple–single queen mating > single–double queen mating. Furthermore, the number of spermatozoa produced per male decreases significantly with reduction in paternity frequency for each species. In contrast, neither sperm length nor male size was significantly associated with the mating system classes or the number of patrilines.Our measures of sperm number provide the first direct evidence that sperm production covaries with the level of sperm competition in a eusocial insect. Given the reversal from multiple to single mating inCataglyphis, our comparative analysis also shows convincingly that reduction in sperm competition influences sperm traits.

Postcopulatory Sexual Selection Generates Speciation Phenotypes in Drosophila

Identifying traits that reproductively isolate species, and the selective forces underlying their divergence, is a central goal of evolutionary biology and speciation research. There is growing recognition that postcopulatory sexual selection, which can drive rapid diversification of interacting ejaculate and female reproductive tract traits that mediate sperm competition, may be an engine of speciation. Conspecific sperm precedence (CSP) is a taxonomically widespread form of reproductive isolation, but the selective causes and divergent traits responsible for CSP are poorly understood. To test the hypothesis that postcopulatory sexual selection can generate reproductive isolation, we expressed GFP or RFP in sperm heads of recently diverged sister species, Drosophila simulans and D. mauritiana, to enable detailed resolution of species-specific sperm precedence mechanisms. Between-species divergence in sperm competition traits and mechanisms prompted six a priori predictions regarding mechanisms of CSP and degree of cross asymmetry in reproductive isolation. We resolved four distinct mechanisms of CSP that were highly consistent with predictions. These comprise interactions between multiple sex-specific traits, including two independent mechanisms by which females exert sophisticated control over sperm fate to favor the conspecific male. Our results confirm that reproductive isolation can quickly arise from diversifying (allopatric) postcopulatory sexual selection. This experimental approach to "speciation phenotypes" illustrates how knowledge of sperm precedence mechanisms can be used to predict the mechanisms and extent of reproductive isolation between populations and species.

Sperm Storage and Sperm Competition in the <I>Helicoverpa armigera</I> (Lepidoptera: Noctuidae)

The mechanism of reproductive behavior and sperm competition strategy in Helicoverpa armigera (Hübner) has been an interesting topic for quite some time. In the current study, sperm motility pattern and sperm competition in H. armigera were investigated. The majority ofapyrene and eupyrene sperm from spermatophore were simultaneously transferred to spermatheca 2 h after copulation. During 3-24 h after copulation, the amounts of apyrene and eupyrene sperm both sharply decreased and only approximately one seventh of one ejaculate remained stable. We used highly variable microsatellite loci to survey 13 families with a total of 379 offspring (mean 29.2 per family). In 8 of 13 families, all offspring were assigned to the last male, and 85.9% of offspring were sired by the last male in total. The possible mechanism of sperm competition in H. armigera encompassed the combined effects of both relatively fixed volume of sperm storage by females and the displacement of sperm from the formerly mated males. This study provides insight into the mechanism underlying sperm precedence in H. armigera, and it is important for potential control of the cotton bollworm.

Laying-order effects on sperm numbers and on paternity: comparing three passerine birds with different life histories

In birds, the number of sperm trapped between the perivitelline membranes around the ovum is an estimate of sperm numbers present at the time and place of fertilisation in the female reproductive tract. Sperm numbers may vary among species and between eggs in a clutch and can provide information about sperm utilisation and mechanisms of sperm competition. Here, we describe patterns of variation in sperm numbers through the egg-laying sequence in three passerines in which extra-pair paternity is common, but copulation behaviour differs. Sperm numbers showed no systematic change across the laying sequence in blue tits (Cyanistes caeruleus), but decreased significantly with laying order in bluethroat (Luscinia svecica) and tree swallow (Tachycineta bicolor) clutches. This is consistent with observations that blue tits regularly copulate throughout the laying sequence, while bluethroats stop mate guarding and tree swallows reduce their copulation frequency once the first egg is laid. Nevertheless, cases of a sudden increase in sperm numbers in clutches of bluethroats and tree swallows suggest that successful inseminations also occurred after laying started. In blue tits and bluethroats, sperm numbers were not higher on extra-pair sired eggs than on eggs sired by the social male, suggesting that extra-pair copulations are not timed to the period of peak fertility for each egg. More extra-pair offspring originated from eggs laid early in the sequence in blue tits, while there was no systematic bias in bluethroats. Our results suggest that copulations during the laying sequence are predominantly performed by within-pair males in our study species.

Resolving variation in the reproductive tradeoff between sperm size and number

Spermatozoa are amongst the most variable cells, and three factors are thought to account for this variation in design: fertilization mode, phylogeny, and postcopulatory sexual selection. In addition, it has long been assumed that a tradeoff exists between sperm size and number, and although postcopulatory sexual selection affects both traits, empirical evidence for a tradeoff has so far been elusive. Our recent theoretical model predicts that the nature of a direct tradeoff between sperm size and number varies with sperm competition mechanism and sperm competition risk. We test these predictions using a comparative approach in two very different taxa with different sperm competition mechanisms: passerine birds (mechanism: simple raffle) and Drosophila fruit flies (sperm displacement). We show that in both groups, males increase their total ejaculate investment with increasing sperm competition risk, but whereas passerine birds allocate disproportionately to sperm number, drosophilids allocate disproportionately to sperm size. This striking difference between the two groups can be at least partly explained by sperm competition mechanisms depending on sperm size relative to the size of the female reproductive tract: in large animals (passerines), sperm numbers are advantageous in sperm competition owing to dilution inside the female tract, whereas in small animals (drosophilids), large sperm are advantageous for physical competition (sperm displacement). Our study provides two important results. First, we provide convincing evidence for the existence of a sperm size-number tradeoff. Second, we show that by considering both sperm competition mechanism and dilution, can we account for variation in sperm size between different taxa.

The strength of postcopulatory sexual selection within natural populations of field crickets

Sperm competition and cryptic female choice are likely to exert strong postcopulatory sexual selection and may amplify or ameliorate selection acting via male mating success. However, relatively few studies have estimated the potential strength of postcopulatory sexual selection acting within natural populations. Field crickets in the genus Teleogryllus have been used extensively as laboratory models to study the evolution of polyandry, and the mechanisms of sperm competition and cryptic female choice. However, we have no knowledge of the extent of multiple mating or patterns of sperm utilization in natural populations of these crickets. We used microsatellite loci to examine the strength of postcopulatory sexual selection in Teleogryllus oceanicus and T. commodus. Genotyping of sperm within sperm storage organs revealed high levels of multiple mating by females in both species, but females utilized sperm from just 75% and 60% of their mates, respectively. Paternity was skewed toward a subset of fathers; skew was strongest in T. oceanicus and declined with the number of fathers represented in both species. Thus, postcopulatory sexual selection is strong in natural populations of both species and must be taken into account when estimating total sexual selection acting on these species. More generally, we argue that more effort should be made to measure the strength of postcopulatory sexual selection in other species, and to integrate episodes of pre- and postcopulatory selection, if we are to gain an accurate appreciation of the evolutionary consequences of sexual selection. Key words: cryptic female choice, paternity skew, polyandry, sperm competition, Teleogryllus commodus, Teleogryllus oceanicus. [Behav Ecol]

Sperm competition and ejaculate economics

Sperm competition was identified in 1970 as a pervasive selective force in post-copulatory sexual selection that occurs when the ejaculates of different males compete to fertilise a given set of ova. Since then, sperm competition has been much studied both empirically and theoretically. Because sperm competition often favours large ejaculates, an important challenge has been to understand the evolution of strategies through which males invest in sperm production and economise sperm allocation to maximise reproductive success under competitive conditions. Sperm competition mechanisms vary greatly, depending on many factors including the level of sperm competition, space constraints in the sperm competition arena, male mating roles, and female influences on sperm utilisation. Consequently, theoretical models of ejaculate economics are complex and varied, often with apparently conflicting predictions. The goal of this review is to synthesise the theoretical basis of ejaculate economics under sperm competition, aiming to provide empiricists with categorised model assumptions and predictions. We show that apparent contradictions between older and newer models can often be reconciled and there is considerable consensus in the predictions generated by different models. We also discuss qualitative empirical support for some of these predictions, and detail quantitative matches between predictions and observations that exist in the yellow dung fly. We argue that ejaculate economic theory represents a powerful heuristic to explain the diversity in ejaculate traits at multiple levels: across species, across males and within individual males. Future progress requires greater understanding of sperm competition mechanisms, quantification of trade-offs between ejaculate allocation and numbers of matings gained, further knowledge of mechanisms of female sperm selection and their associated costs, further investigation of non-sperm ejaculate effects, and theoretical integration of pre- and post-copulatory episodes of sexual selection.

Sperm competition games: Sperm size (mass) and number under raffle and displacement, and the evolution of P2

We examine models for evolution of sperm size (i.e. mass m) and number ( s) under three mechanisms of sperm competition at low ‘risk’ levels: (i) raffle with no constraint on space available for competing sperm, (ii) direct displacement mainly by seminal fluid, and (iii) direct displacement mainly by sperm mass. Increasing sperm mass increases a sperm's ‘competitive weight’ against rival sperm through a diminishing returns function, r( m). ESS total ejaculate expenditure (the product m ⁎ s ⁎) increases in all three models with sperm competition risk, q. If r( m), or ratio r′( m)/ r( m), is independent of ESS sperm numbers, ESS sperm mass remains constant, and the sperm mass/number ratio ( m ⁎/ s ⁎) therefore decreases with risk. Dependency of sperm mass on risk can arise if r( m) depends on competing sperm density (sperm number / space available for sperm competition). Such dependencies generate complex relationships between sperm mass and number with risk, depending both on the mechanism and how sperm density affects r( m). While numbers always increase with risk, mass can either increase or decrease, but m ⁎/ s ⁎ typically decreases with risk unless sperm density strongly influences r( m). Where there is no extrinsic loading due to mating order, ESS paternity of the second (i.e. last) male to mate ( P 2) under displacement always exceeds 0.5, and increases with risk (in the raffle P 2=0.5). Caution is needed when seeking evidence for a sperm size–number trade off. Although size and number trade-off independently against effort spent on acquiring matings, their product, m ⁎ s ⁎, is invariant or fixed at a given risk level, effectively generating a size–number trade off. However, unless controlled for the effects of risk, the relation between m ⁎ and s ⁎ can be either positive or negative (a positive relation is usually taken as evidence against a size–number trade off).

Thiotepa, a reliable marker for sperm precedence measurement in a polyandrous moth

The determination of paternity of offspring produced by polyandrous females is essential for the understanding of sperm competition mechanisms. The sterile male technique using radiation is one of the most commonly employed methods for this purpose. However, sterilization using radiation is likely to be restricted by the equipment availability and cost. Chemosterilization may thus be a cheaper and easier alternative for sterilizing male insects in sperm competition studies. Here we report a reliable chemomarker, thiotepa (N,N′,N″-triethylenethiophosphoramide), for the study of sperm competition and precedence in a polyandrous moth, Ephestia kuehniella (Lepidoptera: Pyralidae). Dipping heads of male moths in 1% thiotepa aqueous solution for 10s resulted in complete sterilization, i.e. their sperm still fertilized eggs but those eggs did not hatch. The sterilization treatment did not significantly affect male copulation ability, female fecundity, and sperm transfer, motility and fertilization. Our results indicate that 86% offspring of the twice-copulated females were fathered by the second males and 14% by the first males. Males treated with 5% thiotepa aqueous solution died within 24h while those treated with 0.5% thiotepa were not fully sterilized.