Sex pheromone component ratios and mating isolation among three Lygus plant bug species of North America

The plant bugs Lygus hesperus, Lygus lineolaris, and Lygus elisus (Hemiptera: Miridae) are major pests of many agricultural crops in North America. Previous studies suggested that females release a sex pheromone attractive to males. Other studies showed that males and females contain microgram amounts of (E)-4-oxo-2-hexenal, hexyl butyrate, and (E)-2-hexenyl butyrate that are emitted as a defense against predators. Using gas chromatography–mass spectrometry, we found that female L. lineolaris and L. elisus have a 4:10 ratio of hexyl butyrate to (E)-2-hexenyl butyrate that is reversed from the 10:1 ratio in female L. hesperus (males of the three species have ~10:1 ratio). These reversed ratios among females of the species suggest a behavioral role. Because both sexes have nearly equal amounts of the major volatiles, females should release more to attract males. This expectation was supported because L. hesperus females released more hexyl butyrate (mean of 86 ng/h) during the night (1800–0700 hours) than did males (<1 ng/h). We used slow-rotating pairs of traps to test the attraction of species to blends of the volatiles with a subtractive method to detect synergism. Each species’ major butyrate ester was released at 3 μg/h, the minor butyrate according to its ratio, and (E)-4-oxo-2-hexenal at 2 μg/h. The resulting catches of only Lygus males suggest that (E)-4-oxo-2-hexenal is an essential sex pheromone component for all three species, (E)-2-hexenyl butyrate is essential for L. elisus and L. lineolaris, and hexyl butyrate is essential for L. hesperus. However, all three components are recognized by each species since ratios of the butyrate esters are critical for conspecific attraction and heterospecific avoidance by males and thus play a role in reproductive isolation among the three species. Because L. hesperus males and females are known to emit these major volatiles for repelling ant predators, our study links defensive allomones in Lygus bugs with an additional use as sex pheromones.     

Juvenile hormone–dopamine systems for the promotion of flight activity in males of the large carpenter bee Xylocopa appendiculata

The reproductive roles of dopamine and dopamine regulation systems are known in social hymenopterans, but the knowledge on the regulation systems in solitary species is still needed. To test the possibility that juvenile hormone (JH) and brain dopamine interact to trigger territorial flight behavior in males of a solitary bee species, the effects on biogenic amines of JH analog treatments and behavioral assays with dopamine injections in males of the large carpenter bee Xylocopa appendiculata were quantified. Brain dopamine levels were significantly higher in methoprene-treated males than in control males 4 days after treatment, but were not significantly different after 7 days. Brain octopamine and serotonin levels did not differ between methoprene-treated and control males at 4 and 7 days after treatment. Injection of dopamine caused significantly higher locomotor activities and a shorter duration for flight initiation in experimental versus control males. These results suggest that brain dopamine can be regulated by JH and enhances flight activities in males. The JH–dopamine system in males of this solitary bee species is similar to that of males of the highly eusocial honeybee Apis mellifera.     

Differential distribution of sperm subpopulations and incidence of pleiomorphisms in ejaculates of captive howling monkeys (Alouatta caraya)

The aim of this study was to develop an objective method to determine the incidence of pleiomorphisms and its influence on the distribution of sperm morphometric subpopulations in ejaculates of howling monkeys (Alouatta caraya) by using a combination of computerized analysis system (ASMA) and principal component analysis (PCA) methods. Ejaculates were collected by electroejaculation methods on a regular basis from five individuals maintained under identical captive environmental, nutritional, and management conditions. Each sperm head was measured for dimensional parameters (Area [A, (square micrometers)], Perimeter [P, (micrometers)], Length [L, (micrometers)], and Width [W, (micrometers)]) and shape-derived parameters (Ellipticity [(L/W)], Elongation [(L???W)/(L?+?W)], and Rugosity [(4лA/P ²)]). PCA revealed two principal components explaining more than the 96 % of the variance. Clustering methods and discriminant analyzes were performed and seven separate subpopulations were identified. There were differences (P?<?0.001) in the distribution of the seven subpopulations as well as in the incidence of abnormal pleiomorphisms (58.6 %, 49.8 %, 35.1 %, 66.4 %, and 55.1 %, P?<?0.05) among the five donors tested. Our results indicated that differences among individuals related to the incidence of pleiomorphisms, and sperm subpopulational structure was not related to the captivity conditions or the sperm collection method, since all individuals were studied under identical conditions. In conclusion, the combination of ASMA and PCA is a useful clinical diagnostic resource for detecting deficiencies in sperm morphology and sperm subpopulations in A. caraya ejaculates that could be used in ex situ conservation programs of threatened species in Alouatta genus or even other endangered neotropical primate species.     

Copulatory behavior in a pholcid spider: males use specialized genitalic movements for sperm removal and copulatory courtship

Sexual selection may operate on pre-copulatory, copulatory, and post-copulatory traits. An example of a copulatory target of sexual selection is the genitalic movements a male performs during copulation. These movements may function either to prevent sperm competition or to influence a female’s fertilization decision. Here we investigated how copulation duration, pedipalp movements, and abdominal movements that males of the pholcid spider Holocnemus pluchei produce during copulation influence sperm removal and/or patterns of successful sperm transfer. We compared mating events with virgin and mated females for differences in copulatory and post-copulatory behavior. We expected longer copulation duration, longer pedipalp movement duration, and more complex and frequent pedipalp and abdominal movements when males mated with mated females compared to virgin females. Except for abdominal movements, our results corroborated these predictions. Furthermore, when we investigated mating events with mated females, we observed sperm mass ejection from the female gonopore and physical removal of sperm by males’ procursi. Females with interrupted second mating events showed a significant reduction of stored sperm masses compared to females with completed mating events. We suggest that males use alternating pedipalp movements to remove most of the rival sperm stored by mated females prior to sperm transfer. Copulation duration and pedipalp movements can be further used to transfer sperm and/or as a form of genitalic copulatory courtship.     

Insights into the role of age and social interactions on the sexual attractiveness of queens in an eusocial bee, <Emphasis Type="Italic">Melipona flavolineata</Emphasis> (Apidae,Meliponini)

The attraction of sexual partners is a vital necessity among insects, and it involves conflict of interests and complex communication systems among male and female. In this study, we investigated the developing of sexual attractiveness in virgin queens (i.e., gynes) of Melipona flavolineata, an eusocial stingless bee. We followed the development of sexual attractiveness in 64 gynes, belonging to seven age classes (0, 3, 6, 9, 15, 18 days post-emergence), and we also evaluated the effect of different social interactions (such as competition between queens and interactions with workers) on the development of attractiveness in other 60 gynes. We used the number of males that tried to mate with a focal gyne as a representative variable of its sexual attractiveness. During the essays, each gyne was individually presented to 10 sexually mature males, and during 3 min, we counted the number of males that everted their genitalia in response to the presence of a gyne. Here, we show that M. flavolineata gynes are capable to (i) maintain their sexual attractiveness for long periods through adult life, (ii) they need a minimum social interaction to trigger the development of sexual attractiveness, and (iii) that gynes express this trait only within a social context. We conclude that the effective occurrence of matings is conditional on potential social interactions that gynes experienced before taking the nuptial flight, when they are still in the nest. These findings bring insights into the factors determining reproductive success in social insects.     

Procurement of exogenous ammonia by the swallowtail butterfly, Papilio polytes, for protein biosynthesis and sperm production

How to acquire sufficient quantity of nitrogen is a pivotal issue for herbivores, particularly for lepidopterans (butterflies and moths) of which diet quality greatly differs among their life stages. Male Lepidoptera often feed from mud puddles, dung, and carrion, a behavior known as puddling, which is thought to be supplementary feeding targeted chiefly at sodium. During copulation, males transfer a spermatophore to females that contains, besides sperm, nutrients (nuptial gifts) rich in sodium, proteins, and amino acids. However, it is still poorly understood how adults, mostly nectarivores, extract nitrogen from the environment. We examined the availability of two ubiquitous inorganic nitrogenous ions in nature, viz. ammonium (or ammonia) and nitrate ions, as nutrients in a butterfly, and show that exogenous ammonia ingested by adult males of the swallowtail, Papilio polytes, can serve as a resource for protein biosynthesis. Feeding experiments with ¹⁵N-labeled ammonium chloride revealed that nitrogen was incorporated into eupyrene spermatozoa, seminal protein, and thoracic muscle. Ammonia uptake by males significantly increased the number of eupyrene sperms in the reproductive tract tissues. The females also had the capacity to assimilate ammonia into egg protein. Consequently, it is evident that acquired ammonia is utilized for the replenishment of proteins allocable for reproduction and somatic maintenance. The active exploitation of exogenous ammonia as a nutrient by a butterfly would foster better understanding of the foraging and reproductive strategies in insects.     

Three-year lifecycle,large body,and very high threshold temperature in the cricket <Emphasis Type="Italic">Gryllus argenteus</Emphasis> for special adaptation to desiccation cycle in Malawi

In temperate climates, the initiation and termination of diapause synchronize the stress-tolerant stage with the stressful season and reproduction with the non-stressful season in many insects. Synchronization is often regulated by photoperiodism.Voltinism and the ultimate size of adults are also important determinants for their lifecycle, and different diapause stages and voltinism patterns are known in crickets.Here, we investigated the life history of the African cricket Gryllus argenteus from Malawi, which is a typical arid tropical highland. The climate is characterized by alternating arid and wet seasons, each of which lasts for half a year, and where the available heat mass is much less than lowlands at the same latitude. We first measured the nymphal duration at each rearing temperature and calculated the lower developmental threshold (t ₀) to be 20.19 °C based on Ikemoto and Takai (2000) and 19.38 °C based on a conventional line-fitting method. These values are very high relative to many other insects. The local temperature in winter does not fall below 15 °C, but this is much higher than the lethal limit. This suggested that critical stress in this locality was not coldness but low precipitation in winter. We estimated, based both on local temperature change and the Ikemoto and Takai’s t ₀, that G. argenteus required 3 years to complete its lifecycle unlike wet lowland species, where univoltinism or multi-voltinism are commonplace. Photoperiodism was observed in this species, but due to a lag between annual cycles in photoperiod, temperature, and humidity, photoperiodism alone cannot atune their lifecycle with local conditions.Synchronization in this species was achieved by three different adaptations: photoperiodism, high t ₀, and large body size, which give it a long lifecycle. Although the species cannot achieve a univoltine lifecycle because of its high t₀ value, it can escape from dry season by entering diapause at moderate temperatures, probably thereby achieving adaptive synchrony of lifecycle with both favorable and unfavorable seasons. A comparison between a conventional photothermogram and a newly formulated photohydrogram or photohygrogram demonstrates that even though sufficient heat is available, scarcity of water and thus scarcity of foliage should force the cricket to maintain diapause at intermediate temperature. The results suggested that high t ₀, large body size, and multi-ennial lifecycle mutually affect each other and formulate a unique adaptation under such an extreme environment.     

Diel periodicity of pheromone release by females of Planococcus citri and Planococcus ficus and the temporal flight activity of their conspecific males

The diel periodicity of sex pheromone release was monitored in two mealybug species, Planococcus citri and Planococcus ficus (Hemiptera; Pseudococcidae), using sequential SPME/GCMS analysis. A maximal release of 2 ng/h pheromone by 9–12-day-old P. citri females occurred 1–2 h before the beginning of photophase. The highest release of pheromone by P. ficus females was 1–2 ng/2 h of 10–20-day-old females, approximately 2 h after the beginning of photophase. Mating resulted in termination of the pheromone release in both mealybug species. The temporal flight activity of the males was monitored in rearing chambers using pheromone baited delta traps. Males of both P. citri and P. ficus displayed the same flight pattern and began flying at 06:00 hours when the light was turned on, reaching a peak during the first and second hour of the photophase. Our results suggest that other biparental mealybug species display also diel periodicities of maximal pheromone release and response. Direct evaluation of the diel periodicity of the pheromone release by the automatic sequential analysis is convenient and will be very helpful in optimizing the airborne collection and identification of other unknown mealybug pheromones and to study the calling behavior of females. Considering this behavior pattern may help to develop more effective pheromone-based management strategies against mealybugs.     

Male fighting and “territoriality” within colonies of the ant Cardiocondyla venustula

The ant genus Cardiocondyla is characterized by a bizarre male polymorphism with wingless fighter males and winged disperser males. Winged males have been lost convergently in several clades, and in at least one of them, wingless males have evolved mutual tolerance. To better understand the evolutionary pathways of reproductive tactics, we investigated Cardiocondyla venustula, a species, which in a phylogenetic analysis clusters with species with fighting and species with mutually tolerant, wingless males. Wingless males of C. venustula use their strong mandibles to kill freshly eclosed rival males and also engage in short fights with other adult males, but in addition show a novel behavior hitherto not reported from social insect males: they spread out in the natal nest and defend “territories” against other males. Ant males therefore show a much larger variety of reproductive tactics than previously assumed.     

Dynamics of sperm transfer in the ant Leptothorax gredleri

Mating tactics differ remarkably between and within species of social Hymenoptera (bees, wasps, ants) concerning, e.g., mating frequencies, sperm competition, and the degree of male sperm limitation. Although social Hymenoptera might, therefore, potentially be ideal model systems for testing sexual selection theory, the dynamics of mating and sperm transfer have rarely been studied in species other than social bees, and basic information needed to draw conclusions about possible sperm competition and female choice is lacking. We investigated sperm transfer in the ant Leptothorax gredleri, a species in which female sexuals attract males by “female calling.” The analysis of 38 female sexuals fixed immediately or up to 7 days after copulation with a single male each revealed that the sperm is transferred into the female bursa copulatrix embedded in a gelatinous mass, presumably a spermatophore. Sperm cells rapidly start to migrate from the tip of the spermatophore towards the spermatheca, but transfer is drastically slowed down by an extreme constriction of the spermathecal duct, through which sperm cells have to pass virtually one by one. This results in the spermatheca being filled only between one and several hours after mating. During this time, the posterior part of the spermatophore seals the junction between bursa copulatrix and spermathecal duct and prevents sperm loss. The prolonged duration of sperm transfer might allow female sexuals to chose between ejaculates and explain previously reported patterns of single paternity of the offspring of multiply mated queens. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development and growth in synanthropic species: plasticity and constraints

Urbanization poses serious extinction risks, yet some species thrive in urban environments. This may be due to a pronounced developmental plasticity in these taxa, since phenotypically, plastic organisms may better adjust to unpredictable urban food resources. We studied phenotypic plasticity in Nuctenea umbratica, a common European forest and urban vegetation spider. We subjected spiderlings to low (LF), medium (MF) and high (HF) food treatments and documented their growth and developmental trajectories into adulthood. Spiders from the three treatments had comparable numbers of instars and growth ratios, but differed in developmental periods. Longest developing LF spiders (♀?=?390, ♂?=?320 days) had the smallest adults, but MF (♀?=?300, ♂?=?240 days) and HF (♀?=?240, ♂?=?210 days) spiders reached comparable adult sizes through shorter development. While males and females had comparable instar numbers, females had longer development, higher growth ratios, adult sizes and mass; and while males adjusted their moulting to food availability, female moulting depended on specific mass, not food treatment. We discussed the patterns of Nuctenea sex-specific development and compared our results with published data on two other Holarctic urban colonizers (Larinioides sclopetarius, Zygiella x-notata) exhibiting high plasticity and fast generation turn-over. We conclude that despite relatively unconstrained developmental time in the laboratory enabling Nuctenea to achieve maximal mass and size—main female fitness proxies—their relatively fixed growth ratio and long generation turn-over may explain their lower success in urban environments.     

Sperm-depleted males influence the reproductive behaviour of conspecifics

In many insect species, sperm-depleted males (SDMs, i.e. males that have exhausted their sperm after a given number of matings) remain sexually active and continue to mate females. Here, we investigated the behavioural modifications that occur in both sexes of the parasitoid Asobara tabida Nees (Hymenoptera: Braconidae), after matings by fertile males and sperm-depleted males. We show that (i) virgin females, mated females and females mated to a SDM exhibited different behaviours and that (ii) males responded differently to females depending on whether the females had previously mated with an SDM or not. Our findings demonstrate that SDM influenced the reproductive behaviour of both males and females, especially with regard to male responsiveness and female attractiveness. These findings are discussed in the context of adaptive behaviour and fitness maximization in both males and females.     

Inhibition of Murine Norovirus and Feline Calicivirus by Edible Herbal Extracts

Human noroviruses (HuNoVs) cause foodborne and waterborne viral gastroenteritis worldwide. Because HuNoV culture systems have not been developed thus far, no available medicines or vaccines preventing infection with HuNoVs exist. Some herbal extracts were considered as phytomedicines because of their bioactive components. In this study, the inhibitory effects of 29 edible herbal extracts against the norovirus surrogates murine norovirus (MNV) and feline calicivirus (FCV) were examined. FCV was significantly inhibited to 86.89 ± 2.01 and 48.71 ± 7.38% by 100 μg/mL of Camellia sinensis and Ficus carica, respectively. Similarly, ribavirin at a concentration of 100 μM significantly reduced the titer of FCV by 77.69 ± 10.40%. Pleuropterus multiflorus (20 μg/mL) showed antiviral activity of 53.33 ± 5.77, and 50.00 ± 16.67% inhibition was observed after treatment with 20 μg/mL of Alnus japonica. MNV was inhibited with ribavirin by 59.22 ± 16.28% at a concentration of 100 μM. Interestingly, MNV was significantly inhibited with 150 µg/mL Inonotus obliquus and 50 μg/mL Crataegus pinnatifida by 91.67 ± 5.05 and 57.66 ± 3.36%, respectively. Treatment with 20 µg/mL Coriandrum sativum slightly reduced MNV by 45.24 ± 4.12%. The seven herbal extracts of C. sinensis, F. carica, P. multiflorus, A. japonica, I. obliquus, C. pinnatifida, and C. sativum may have the potential to control noroviruses without cytotoxicity.     

Giant honeybees (Apis dorsata) mob wasps away from the nest by directed visual patterns

The open nesting behaviour of giant honeybees (Apis dorsata) accounts for the evolution of a series of defence strategies to protect the colonies from predation. In particular, the concerted action of shimmering behaviour is known to effectively confuse and repel predators. In shimmering, bees on the nest surface flip their abdomens in a highly coordinated manner to generate Mexican wave-like patterns. The paper documents a further-going capacity of this kind of collective defence: the visual patterns of shimmering waves align regarding their directional characteristics with the projected flight manoeuvres of the wasps when preying in front of the bees’ nest. The honeybees take here advantage of a threefold asymmetry intrinsic to the prey–predator interaction: (a) the visual patterns of shimmering turn faster than the wasps on their flight path, (b) they “follow” the wasps more persistently (up to 100 ms) than the wasps “follow” the shimmering patterns (up to 40 ms) and (c) the shimmering patterns align with the wasps’ flight in all directions at the same strength, whereas the wasps have some preference for horizontal correspondence. The findings give evidence that shimmering honeybees utilize directional alignment to enforce their repelling power against preying wasps. This phenomenon can be identified as predator driving which is generally associated with mobbing behaviour (particularly known in selfish herds of vertebrate species), which is, until now, not reported in insects.     

Volcanic mercury in Pinus canariensis

Mercury (Hg) is a toxic element that is emitted to the atmosphere by both human activities and natural processes. Volcanic emissions are considered a natural source of mercury in the environment. In some cases, tree ring records taken close to volcanoes and their relation to volcanic activity over time are contradictory. In 1949, the Hoyo Negro volcano (La Palma-Canary Islands) produced significant pyroclastic flows that damaged the nearby stand of Pinus canariensis. Recently, 60 years after the eruption, we assessed mercury concentrations in the stem of a pine which survived volcano formation, located at a distance of 50 m from the crater. We show that Hg content in a wound caused by pyroclastic impacts (22.3 μg kg^?1) is an order of magnitude higher than the Hg concentrations measured in the xylem before and after the eruption (2.3 μg kg^?1). Thus, mercury emissions originating from the eruption remained only as a mark—in pyroclastic wounds—and can be considered a sporadic and very high mercury input that did not affect the overall Hg input in the xylem. In addition, mercury contents recorded in the phloem (9.5 μg kg^?1) and bark (6.0 μg kg^?1) suggest that mercury shifts towards non-living tissues of the pine, an aspect that can be related to detoxification in volcanism-adapted species.     

NirS-type denitrifying bacteria in aerobic water layers of two drinking water reservoirs: Insights into the abundance, community diversity and co-existence model

The nirS-type denitrifying bacterial community is the main drivers of the nitrogen loss process in drinking water reservoir ecosystems.The temporal patterns in nirS gene abundance and nirS-type denitrifying bacterial community harbored in aerobic water layers of drinking water reservoirs have not been studied well.In this study,quantitative polymerase chain reaction (qPCR) and Illumina Miseq sequencing were employed to explore the nirS gene abundance and denitrifying bacterial community structur...     

Torpor in free-ranging antechinus: does it increase fitness?

Antechinus are small, insectivorous, heterothermic marsupial mammals that use torpor from late summer to early winter and reproduce once a year in late winter/early spring. Males die after mating, most females produce only a single litter, but some survive a second winter and produce another litter. As it is not known how these females manage to survive the second winter after the energetically demanding reproductive period and then reproduce a second time, we aimed to provide the first data on thermal biology of free-ranging antechinus by using temperature telemetry. Male Antechinus stuartii and Antechinus flavipes rarely entered torpor in autumn/early winter in the wild, expressing only shallow bouts of <2 h. Female A. stuartii used torpor extensively, employing bouts up to 16.7 h with body temperatures as low as 17.8 °C. Interestingly, although first and second year females used similar torpor patterns, torpor occurrence was almost twofold in second year (93 % of days) than first year females (49 %), and the proportion of the overall monitoring period animals spent torpid was 3.2-fold longer in the former with a corresponding shorter activity period. Our study suggests that intensive use of torpor is crucial for second year females for autumn and winter survival and production of a second litter. We provide the first evidence of an age-related pattern in daily torpor expression in free-ranging mammals and show that torpor use is a complex process that is affected not only by the current energy availability and thermal conditions but also by the reproductive history and age of individuals.     

Limited scope for reproductive senescence in wild populations of a short-lived fish

Senescence in wild populations was long considered negligible but current evidence suggests that it is widespread in natural populations of mammals and birds, affecting the survival and reproductive output of older individuals. In contrast, little is known about reproductive senescence in species with asymptotic growth that can keep increasing their reproductive output as they grow older and larger. Using a cross-sectional study, we tested age-related decline in fecundity and relative allocation to reproduction in five wild populations of an annual killifish, Nothobranchius furzeri (Cyprinodontiformes). We did not detect any decline in absolute female egg production over their short lifespan in the wild. Relative fecundity (egg production controlled for female body mass) tended to decrease with age. This effect was driven primarily by a single population that survived 17 weeks, almost twice as long as the median persistence of the other four study populations. There was no decrease in relative ovary mass while in males, relative testes mass actually increased with age. Intra-population variation in relative ovary mass increased in older females suggesting heterogeneity in individual trajectories of female reproductive allocation. Overall, we demonstrate that annual killifish do not experience significant age-related decline in reproductive functions during their very short lifespan in the wild despite the marked deterioration of gonad tissue detected in captivity.     

A male gift to its partner? Cyanogenic glycosides in the spermatophore of longwing butterflies (<Emphasis Type="Italic">Heliconius</Emphasis>)

Males of several insect species transfer nuptial gifts to females during mating, typically in the form of a protein-rich spermatophore. In chemically defended species, males could potentially enhance such a gift with chemicals that help protect the female, her eggs, or both. This was shown for lepidopteran species that accumulate pyrrolizidine alkaloids. Most Heliconius butterflies are presumably protected from predators by virtue of de novo synthesized and/or sequestered cyanogenic glycosides. Males of Heliconius species are known to transfer nutritional gifts to the females but whether defensive chemicals could also be transferred is not known. To ascertain whether transfer of cyanogens occurs, we dissected freshly mated females from nine different Heliconius species and analyzed spermatophores for cyanogenic glycosides. We found cyanogens in the spermatophores of all nine species. This is the first time cyanogenic glycosides are reported in the spermatophores of arthropods. We discuss the implications of these findings for Heliconius biology and for other cyanogenic insects as well. We suggest that chemically defended species commonly lace their nuptial gifts with defensive chemicals to improve gift quality.     

Large- and small-size advantages in sneaking behaviour in the dusky frillgoby <Emphasis Type="Italic">Bathygobius fuscus</Emphasis>

Sneaking tactic, a male alternative reproductive tactic involving sperm competition, is generally adopted by small individuals because of its inconspicuousness. However, large size has an advantage when competition occurs between sneakers for fertilization of eggs. Here, we suggest that both large- and small-size advantages of sneaker males are present within the same species. Large sneaker males of the dusky frillgoby Bathygobius fuscus showed a high success rate in intruding into spawning nests because of their advantage in competition among sneaker males in keeping a suitable position to sneak, whereas small sneakers had few chances to sneak. However, small sneaker males were able to stay in the nests longer than large sneaker males when they succeeded in sneak intrusion. This suggests the possibility of an increase in their paternity. The findings of these size-specific behavioural advantages may be important in considering the evolution of size-related reproductive traits.