Mating Sequence Research Articles

Reproductive output of old males is limited by seminal fluid, not sperm number

Abstract Male reproductive senescence is typically characterized by a decline in the number of sperm produced and transferred by old males, a phenomenon that may be exacerbated in polygynous species where males mate multiply. However, males also transfer seminal fluid to females, and little is known about its role in modulating male reproductive senescence. Here, we explore the contributions of sperm and seminal fluid towards male reproductive senescence in a series of sequential matings, using Drosophila melanogaster. As expected, old males produce fewer offspring than young males. However, this pattern is not driven by sperm limitation: old males have more sperm and transfer similar numbers to females, compared to young males. Instead, females storing fewer sperm of old males compared to that of young males, over a long term, drives male reproductive senescence. We are able to mitigate the age-related decline in male reproductive output by supplementing females with the seminal fluid of a young male, before she mates with an old male. Similarly, we alleviate the reduction in reproductive output across sequential matings by supplementing females with seminal fluid. Our findings highlight that seminal fluid, rather than sperm number, limits reproductive success in old or multiply mating males, highlighting its underappreciated role in reproductive aging.

Don't touch me! Mating not always preceded by courtship behavior in predatory coccinellids.

Knowledge of the ecology and behavior of biological control agents is essential for their effective use in biocontrol and Integrated Pest Management (IPM)programs. There is a lack of information regarding the courtship and mating traits that are crucial for successful mating in biocontrol population of coccinellids. To expand our current understanding in this area, 2 coccinellid species commonly used for the biocontrol of soft-bodied insects, Cryptolaemus montrouzieri (Mulsant) and Propylea quatuordecimpunctata (Linnaeus) (Coleoptera: Coccinellidae), were studied. A quantitative analysis was performed to compare their courtship and mating displays. Key behavioral traits of the precopulatory, copulatory, and postcopulatory phases were investigated. The courtship and mating sequences of C. montrouzieri and P. quatuordecimpunctata were similar to each other. During the mating phase, the key displays were the opening of the elytrae and wings followed by body shaking in C. montrouzieri and leg tapping followed by body shaking in P. quatuordecimpunctata. The mating success of both species was not correlated with any courtship display, such as antennal tapping. Only in C. montrouzieri mounting attempt showed a higher frequency from the backside without affecting male mating success. This research adds baseline knowledge about the courtship and mating behavior of the biocontrol population of coccinellids, contributing to the identification of potentiallyuseful benchmarks (e.g., body shaking, leg tapping) for monitoring prolonged mass-rearing processes, thusreducing mating failures.

Emotional vocalizations alter behaviors and neurochemical release into the amygdala

The basolateral amygdala (BLA), a brain center of emotional expression, contributes to acoustic communication by first interpreting the meaning of social sounds in the context of the listener’s internal state, then organizing the appropriate behavioral responses. We propose that modulatory neurochemicals such as acetylcholine (ACh) and dopamine (DA) provide internal-state signals to the BLA while an animal listens to social vocalizations. We tested this in a vocal playback experiment utilizing highly affective vocal sequences associated with either mating or restraint, then sampled and analyzed fluids within the BLA for a broad range of neurochemicals and observed behavioral responses of adult male and female mice. In male mice, playback of restraint vocalizations increased ACh release and usually decreased DA release, while playback of mating sequences evoked the opposite neurochemical release patterns. In non-estrus female mice, patterns of ACh and DA release with mating playback were similar to males. Estrus females, however, showed increased ACh, associated with vigilance, as well as increased DA, associated with reward-seeking. Experimental groups that showed increased ACh release also showed the largest increases in an aversive behavior. These neurochemical release patterns and several behavioral responses depended on a single prior experience with the mating and restraint behaviors. Our results support a model in which ACh and DA provide contextual information to sound analyzing BLA neurons that modulate their output to downstream brain regions controlling behavioral responses to social vocalizations.

Emotional vocalizations alter behaviors and neurochemical release into the amygdala.

The basolateral amygdala (BLA), a brain center of emotional expression, contributes to acoustic communication by first interpreting the meaning of social sounds in the context of the listener's internal state, then organizing the appropriate behavioral responses. We propose that modulatory neurochemicals such as acetylcholine (ACh) and dopamine (DA) provide internal-state signals to the BLA while an animal listens to social vocalizations. We tested this in a vocal playback experiment utilizing highly affective vocal sequences associated with either mating or restraint, then sampled and analyzed fluids within the BLA for a broad range of neurochemicals and observed behavioral responses of adult male and female mice. In male mice, playback of restraint vocalizations increased ACh release and usually decreased DA release, while playback of mating sequences evoked the opposite neurochemical release patterns. In non-estrus female mice, patterns of ACh and DA release with mating playback were similar to males. Estrus females, however, showed increased ACh, associated with vigilance, as well as increased DA, associated with reward-seeking. Experimental groups that showed increased ACh release also showed the largest increases in an aversive behavior. These neurochemical release patterns and several behavioral responses depended on a single prior experience with the mating and restraint behaviors. Our results support a model in which ACh and DA provide contextual information to sound analyzing BLA neurons that modulate their output to downstream brain regions controlling behavioral responses to social vocalizations.

Experience matters: genetic variation affects male reproductive success across sequential mating events in Drosophila melanogaster.

The cost of reproduction is well studied in females but only recently have the costs of mating been investigated in males. Research suggests that males allocate resources between subsequent mating events, resulting in differential success across mating bouts. Selection should favor allocation strategies that match the likelihood of successive matings. The complexity of the system, however, suggests that one fixed strategy is unlikely to be universally favored and thus I predict that genetic variation for different allocation strategies will be segregating in natural populations. To test this, I measured several components of reproductive performance in eight inbred genotypes of Drosophila melanogaster across three sequential mating events. As predicted, there was genetic variation for how previous experience affected a male's reproductive performance for both the proportion of matings that produced offspring and the proportion of offspring sired (P1). Some genotypes had the highest success in their first matings and declined in successive matings while other genotypes did best in later matings. Mating experience had consistent effects across genotypes on fertility and induced refractoriness to remating. On average, virgin matings produced the highest fertility and third matings most effectively induced refractoriness. Genotype also had a significant effect on fertility. These results have important implications for understanding how selection may be acting on males when there is variation in the likelihood of multiple mating events and could affect the evolution of male allocation strategies in the face of perceived competitors.

Factors Influencing Copulation Duration in Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae).

The gregarious ectoparasitic beetle Dastarcus helophoroides (Fairmaire) is considered a primary biocontrol agent for controlling several cerambycid pests in East Asian countries. A thorough study of reproductive behavior is a prerequisite for the mass production of natural insect predators. Nonetheless, little attention has been given to this ectoparasitic beetle. We performed a series of trials to assess whether the adult copulation duration, a key behavioral trait, is differentially influenced by physiological and ecological factors, including body size, mating history, kinship, sex ratio, mating sequence, feeding status, ambient temperature, photoperiod, and time of day. Additionally, the effect of the copulation duration on the reproductive output of this beetle was also investigated. The results indicated that the copulation duration varied considerably, ranging from 1.12 min to 16.40 min and lasting for an average of 9.11 ± 0.12 min. Females with longer copulations laid more eggs and had a greater proportion of eggs hatched. Medium-sized individuals copulated significantly longer than small- and large-sized individuals. The copulation durations were significantly longer when both sexes experienced an asymmetric mating history than when both sexes experienced a symmetric mating history. Inbred couples copulated significantly longer than outbred couples. In terms of the adult sex ratio, increasing the density of females (polygamous group) or males (polyandrous group) led to significantly longer copulation durations than those in the monogamous group. The copulation durations gradually decreased with increasing the mating sequence and temperature. Food-absence couples copulated significantly longer than food-presence couples. The mean copulation duration of the scotophase was significantly longer than that of the photophase. These results demonstrate that all of the analyzed factors emerge as important factors influencing the copulation duration, ultimately affecting the reproductive outputs in this ectoparasitic beetle.

Long-term Decline of the Spotted Salamander (Ambystoma maculatum) in an Undeveloped Landscape

Abstract The Eastern Hellbender (Cryptobranchus alleganiensis alleganiensis) is a giant, charismatic species of salamander of conservation concern. Despite growing interest in their breeding behavior, significant gaps remain in our understanding of hellbender reproduction, particularly the behavior occurring immediately prior to and during breeding, as these activities typically occur within the nesting cavity and out of view. In this study, we used custom-built infrared cameras installed underwater in artificial nesting shelters to record pre-breeding behaviors, complete mating sequences, and failed mating attempts in eleven shelters. Using these recordings, we describe the basic mating sequence, the presence of potential alternative mating tactics, and two novel behaviors, including possible signal production via wave-based communication and unique egg laying behavior by female hellbenders. These findings add to our understanding of hellbenders’ life history as well as informing conservation efforts in both captive and wild environments.

Lateralized Movements during the Mating Behavior, Which Are Associated with Sex and Sexual Experience, Increase the Mating Success in Alphitobius diaperinus (Coleoptera: Tenebrionidae).

In the present study, we explored the effects of displacement directionality in mating behavior (i.e., lateralized and non-lateralized movements) on mating success (i.e., copulation occurs) and efficiency (i.e., time length at which copulation is achieved), and its association with sex and sexual experience in A. diaperinus. To do so, we carried out mating experiments and recorded the behavior of the mating pair during the whole mating sequence (i.e., precopulatory and copulatory phases). During the precopulatory phase, independently of sex and sexual experience, all beetles performed non-lateralized (i.e., backside or frontside) approaches; however, only sexually experienced beetles showed lateralized approaches (i.e., right-side and left-side). Notably, experienced males exhibited greater mating success than virgin males. After the approach, both virgin and experienced males displayed lateralized and non-lateralized mounts on the females with distinct mating success. Regardless of their sexual experience, 100% of successful mating attempts were achieved when males mounted from the females' right side. Furthermore, the development of lateralized approaches and mounts reduces the time of mating sequence span compared with non-lateralized behaviors. We highlight the importance of lateralization in mating behavior and sexual experience to achieve higher mating success, addressing a potential learning ability of beetles based on experience.

Ethogram and classification of the mating and egg-laying behaviour of the Southeast Asian apple snail Pilavirescens (Deshayes, 1824) (Mollusca, Gastropoda, Caenogastropoda, Ampullariidae).

The status of the indigenous Southeast Asian apple snails belonging to the genus Pila is of concern due to their fast rate of population decline, possibly as a result of multiple factors including habitat loss or disturbance and the introduction of globally-invasive apple snails, Pomacea spp. Conservation actions, including captive breeding of the native Pila species, have been suggested as urgent remedial practices, but the lack of knowledge regarding the fundamental reproductive biology of indigenous Pila spp. makes such practices difficult. In the present study, observations on the mating and egg-laying behaviour of an economic valuable apple snail native to Southeast Asia, P.virescens, were conducted using video recording to examine and describe their reproductive behaviour under a laboratory condition. A total of 15 types of mating and seven egg-laying behaviour were recorded. The mating sequence which subsequently resulted in egg laying was comprised of seven types of major sequential behaviour: mate probing, mounting, shell circling, positioning, insemination posture, sheath withdrawal and dismounting. Rejection of mating attempts by the female was frequently observed. Egg laying occurred during either day or night. A sequence of seven distinct types of behaviour were performed during oviposition: climbing, positioning, forming a temporary tube, mucous secreting, egg depositing, leaving and resting. Overall, these results provide an understanding of the egg-laying behavioural process and highlight its complexity in P.virescens. In addition, detailed ethograms of mating and egg-laying behaviour were derived. These will promote further systematic comparative studies of the reproductive behaviour of apple snails.

Being exposed to Acmella oleracea-based insecticide extract reduces mobility and mating success in Prostephanus truncatus, the major pest of maize in storages

The evaluation of sublethal effects of botanical insecticides is a key tool to develop new strategies in pest management programs, since they potentially affect insect physiology and behavior. Acmella oleracea (L.) R.K. Jansen (Asteraceae) is a medicinal food plant widely used on an industrial level and with recognized efficacy against various arthropod pests and vectors. In this study, the sublethal effects of an A. oleracea hexane extract (HE) on the mobility, mating, and behavior of Prostephanus truncatus were investigated. The concentrations that killed 10, 30 and 50% of the exposed adults were 7.01, 19.5, and 39.5 mg/mL, respectively. The walking behavioral traits, i.e., duration and number of stops, and upside down (i.e., the time spent by the beetle on its back), were significantly higher in the adults exposed to LC30 than LC10 and control ones. The highest velocity was recorded for control beetles (21.5 mm/s), followed by LC10- (20.7 mm/s) and LC30-treated ones (21.2 mm/s). Concerning lateralization, P. truncatus males were right biased during the mating sequence. Sublethal concentrations negatively affected the mating success of LC10- and LC30-exposed males in comparison with control ones. Overall, our findings outlined that the exposure of P. truncatus adults to A. oleracea HE LC10 and LC30 deeply affected their walking and mating behavior, a fact that should be considered further when optimizing pest management tactics in storages.

How aggressive interactions with biomimetic agents optimize reproductive performances in mass-reared males of the Mediterranean fruit fly

Mass-rearing procedures of insect species, often used in biological control and Sterile Insect Technique, can reduce the insects competitiveness in foraging, dispersal, and mating. The evocation of certain behaviours responsible to induce specific neuroendocrine products may restore or improve the competitiveness of mass-reared individuals. Herein, we used a mass-reared strain of Ceratitis capitata as model organism. C. capitata is a polyphagous pest exhibiting territorial displays that are closely related to its reproductive performance. We tested if the behaviour of C. capitata males could be altered by hybrid aggressive interactions with a conspecific-mimicking robotic fly, leading to more competitive individuals in subsequent mating events. Aggressive interactions with the robotic fly had a notable effect on subsequent courtship and mating sequences of males that performed longer courtship displays compared to naïve individuals. Furthermore, previous interactions with the robotic fly produced a higher mating success of males. Reproductive performances of C. capitata males may be improved by specific octopaminergic neurones activated during previous aggressive interactions with the robotic fly. This study adds fundamental knowledge on the potential role of specific neuro-behavioural processes in the ecology of tephritid species and paves the way to innovative biotechnological control methods based on robotics and bionics.

Effect of Ageing in the Mating Behaviour Sequence of Osmia cornuta Latr. (Hymenoptera: Megachilidae).

Osmia cornuta Latr. is largely managed worldwide for the pollination of orchard crops, playing a key role in the maintenance of healthy ecosystems and ensuring economic and social benefits for human society. The management techniques of this pollinator include the possibility of delaying emergence from cocoons after diapause, allowing for the pollination of later-blooming fruit crops. In this study, the mating behaviour of bees emerging at the natural time (Right Emergence Insects) and of late-emerged bees (Aged Emergence Insects) was described in order to test if a delay in emergence could affect the mating sequence of O. cornuta. Markov analysis of the mating behaviour revealed the occurrence of antenna motion episodes that were repeated in a stereotyped manner at regular intervals during the mating sequence of both Right Emergence Insects and in Aged Emergence Insects. Pouncing, rhythmic and continuous emission of sound, motion of antennae, stretching of the abdomen, short and long copulations, scratching, inactivity, and self-grooming were identified as the stereotyped behavioural units of a behavioural sequence. The occurrence of short copulations, the frequency of which increased with the age of bees, could lead to a failure in the reproduction of the mason bee.

Ranking parameters driving siring success during sperm competition in the North African houbara bustard

Sperm competition is a powerful force driving the evolution of ejaculate and sperm traits. However, the outcome of sperm competition depends on many traits that extend beyond ejaculate quality. Here, we study male North African houbara bustards (Chlamydotis undulata undulata) competing for egg fertilization, after artificial insemination, with the aim to rank the importance of 14 parameters as drivers of siring success. Using a machine learning approach, we show that traits independent of male quality (i.e., insemination order, delay between insemination and egg laying) are the most important predictors of siring success. Traits describing intrinsic male quality (i.e., number of sperm in the ejaculate, mass motility index) are also positively associated with siring success, but their contribution to explaining the outcome of sperm competition is much lower than for insemination order. Overall, this analysis shows that males mating at the last position in the mating sequence have the best chance to win the competition for egg fertilization. This raises the question of the importance of female behavior as determinant of mating order.

Sexual adaptation: is female–male mounting a supernormal courtship display in Japanese macaques?

Abstract We analysed heterosexual consortships in a free-ranging group of Japanese macaques in which adult females routinely perform female-to-male mounting (FMM). We tested whether FMM is more efficient (i.e., a ‘supernormal courtship’ behavioural pattern) than species-typical female-to-male sexual solicitations (FMSS) at prompting subsequent male-to-female mounts (MFM). In a context of high female-female competition for male mates, we found that (1) FMM functioned to focus the male consort partner’s attention as efficiently as FMSS and prevented him from moving away, and (2) FMM was more efficient than species-typical FMSS at expediting MFM (i.e., the most fitness-enhancing sexual behaviour of a mating sequence). We concluded that FMM could be considered a supernormal courtship behavioural pattern in adult female Japanese macaques. This population-specific sexual adaptation may result from a combination of favourable socio-demographic conditions. This study has implications for the evolutionary history of non-conceptive mounting patterns in Japanese macaques and non-conceptive sexuality in humans.

Effects of Irradiated Sterile Male and Mating Sequence on the Fertility of Drosophila suzukii (Diptera: Drosophilidae).

The sterile insect technique has been explored in the laboratory to control populations of Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), a globally invasive pest. We studied the reproductive behavior of D. suzukii including mating frequency, time between matings, and mating duration among non-irradiated flies. Irradiation doses were tested at 0, 60, 90, 110, 120, 150, and 180 Gy to select the optimal dose for producing sterile males. In addition, we examined the effects of mating sequence on offspring production where females were presented with irradiated males first and then wild males, or the reverse. Female D. suzukii were found to mate twice on average through their lifespan, with 16.53 ± 12.05 d between matings. The first mating duration was 24.64 ± 1.52 min shorter than the second mating. A dose of 90 Gy was suitable where irradiated males lived as long as non-irradiated males, and few eggs hatched from matings. The mating sequence experiment revealed first-male parentage preference. Wild females that mated with a wild male and then irradiated male produced more offspring than females mated with an irradiated and then wild male. Overall, the influence of mating sequence should be taken into consideration when applying the sterile insect technique (SIT) to control D. suzukii populations.

A preliminary study to identify and distinguish southern tropical populations of Ganoderma boninense from oil palm via mating assays, sequence data, and microsatellite markers

ABSTRACT Morphological studies suggest that the major pathogen causing basal stem rot of oil palm in Papua New Guinea and Solomon Islands is Ganoderma boninense. This study presents the first evidence for conspecificity of G. boninense from four countries where basal stem rot is prevalent. Seventy-three dikaryotic isolates of Ganoderma boninense from Indonesia, Malaysia, Papua New Guinea, and Solomon Islands were studied via mating tests, analyses of nuc internal transcribed spacer ITS1-5.8S-ITS2 sequences, and microsatellite genotyping. Sequence similarity in the ITS1-5.8S-ITS2 region was >99%, and all exotic isolates successfully mated with Papua New Guinea tester strains. Transfer of nuclei during mating was also confirmed via microsatellite markers for the first time in this species. Four microsatellite primers were used to generate evidence for 33 alleles in the four populations. All isolates studied had unique genetic fingerprints but alleles were also shared, suggesting gene flow. Heterozygosities were lower than expected in Indonesian and Papua New Guinea populations, consistent with the possibility of localized inbreeding.

Behavioral Asymmetries Affecting Male Mating Success in Tenebrio molitor (Coleoptera: Tenebrionidae), an Important Edible Species

The yellow mealworm, Tenebrio molitor L., is one of the most significant insect species of economic importance for producing protein-rich food and feed. The larvae are a promising fishmeal substitute for fish feed, and a good alternative source for human nutrition. In this study, the effect of behavioral asymmetries on male mating success of T. molitor was evaluated. Males performing antennal waving (63%) when detecting a female approached the apex of the female abdomen in a comparable manner from both sides (32% from left side vs 31% from right side). Fewer males showed antennal waving and also raised the anterior part of their body (37%) during mate recognition; 14% of them approached on the apex of female abdomen from the left side, and 23% of them approached from the right side of female body. The duration of mate recognition, antennal tapping, rubbing behavior, and the whole mating sequence of males that moved on the apex of the abdomen from the left side of females was significantly lower over that of males approaching from the right side (10.7, 12.6, 16.4, and 126.1 s for left-biased males vs 12.2, 14.8, 18.6, and 139.6 s for right-biased males, respectively). Concerning mounting side, left-biased males showed a shorter duration of rubbing behavior and the whole mating sequence (15.5 and 123.2 s) over right-biased (18.9 and 138.3 s) and backside approaching males (19.4 and 144.1 s). The duration of mate recognition, antennal tapping, and copula was not affected by the presence of a male laterality bias. Overall, this study sheds light on how laterality affects mating traits and the male success of this important edible insect species.

Improved dairy cattle mating plans at herd level using genomic information

From 2012 to 2018, 223 180 Montbéliarde females were genotyped in France and the number of newly genotyped females increased at a rate of about 33% each year. With female genotyping information, farmers have access to the genomic estimated breeding values of the females in their herd and to their carrier status for genetic defects or major genes segregating in the breed. This information, combined with genomic coancestry, can be used when planning matings in order to maximize the expected on-farm profit of future female offspring. We compared different mating allocation approaches for their capacity to maximize the expected genetic gain while limiting expected progeny inbreeding and the probability to conceive an offspring homozygous for a lethal recessive allele. Three mate allocation strategies (random mating (RAND), sequential mating (gSEQ€) and linear programing mating (gLP€)) were compared on 160 actual Montbéliarde herds using male and female genomic information. Then, we assessed the benefit of using female genomic information by comparing matings planned using only female pedigree information with the equivalent strategy using genomic information. We measured the benefit of adding genomic expected inbreeding and risk of conception of an offspring homozygous for a lethal recessive allele to Net merit in mating plans. The influence of three constraints was tested: by relaxing the constraint on availability of a particular semen type (sexed or conventional) for bulls, by adding an upper limit of 8.5% coancestry between mate pairs or by using a more stringent maximum use of a bull in a herd (5% vs 10%). The use of genomic information instead of pedigree information improved the mate allocation method in terms of progeny expected genetic merit, genetic diversity and risk to conceive an offspring homozygous for a lethal recessive allele. Optimizing mate allocation using linear programming and constraining coancestry to a maximum of 8.5% per mate pair reduced the average coancestry with a small impact on expected Net Merit. In summary, for male and female selection pathways, using genomic information is more efficient than using pedigree information to maximize genetic gain while constraining the expected inbreeding of the progeny and the risk to conceive an offspring homozygous for a lethal recessive allele. This study also underlines the key role of semen type (sexed vs conventional) and the associated constraints on the mate allocation algorithm to maximize genetic gain while maintaining genetic diversity and limiting the risk to conceive an offspring homozygous for a lethal recessive allele.

Direct glia-to-neuron transdifferentiation gives rise to a pair of male-specific neurons that ensure nimble male mating.

Sexually dimorphic behaviours require underlying differences in the nervous system between males and females. The extent to which nervous systems are sexually dimorphic and the cellular and molecular mechanisms that regulate these differences are only beginning to be understood. We reveal here a novel mechanism by which male-specific neurons are generated in Caenorhabditis elegans through the direct transdifferentiation of sex-shared glial cells. This glia-to-neuron cell fate switch occurs during male sexual maturation under the cell-autonomous control of the sex-determination pathway. We show that the neurons generated are cholinergic, peptidergic, and ciliated putative proprioceptors which integrate into male-specific circuits for copulation. These neurons ensure coordinated backward movement along the mate's body during mating. One step of the mating sequence regulated by these neurons is an alternative readjustment movement performed when intromission becomes difficult to achieve. Our findings reveal programmed transdifferentiation as a developmental mechanism underlying flexibility in innate behaviour.

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.