The absence of an established in vitro platform is a major obstacle in research on parasitic flatworms, including Fasciola flukes. Fasciola flukes cause zoonotic infections that primarily affect the liver and the bile ducts. Infected juveniles can cause severe liver damage in animals, occasionally leading to sudden death. Although resistance to the only drug for the acute liver stage has been reported worldwide, the search for new drugs has been unsuccessful owing to the critical limitations of previous in vitro cultures. Previous studies have been unable to reproduce liver-stage development in vitro, hindering research on this stage. This study aimed to provide a novel in vitro research platform using a laboratory strain of Fasciola hepatica/gigantica hybrid. Juveniles derived from the livers of mice at 7 and 11 days post-infection (dpi) survived for nearly 100 days in the basic medium consisting of Roswell Park Memorial Institute (RPMI) 1640 supplemented with 50% fetal bovine serum. Bovine red blood cells (RBC) and sex-inducing substances (SIS) that induce sexualization in a free-living flatworm (planarian) were supplemented to examine their effects on the developmental processes in the liver stage, including growth, body shape change, and reproductive development. SIS induced all three processes, although the last was incomplete, suggesting that the sex-inducing ability of SIS is conserved between free-living and parasitic flatworms. However, RBC was somewhat toxic and less effective than SIS for both growth and reproductive development and could not alter body shape. Furthermore, the combined effects of the two supplements were not observed. In this study, the reproducibility of the development was carefully confirmed, and it was shown that a single SIS supplementation is currently the best condition and more closely mimics liver-stage development. This study provides a preliminary but outstanding in vitro research platform for liver-stage juveniles and will facilitate further drug development.

Many metazoans switch between asexual and sexual reproduction based on environmental changes, life cycle phases, or both. This reproductive strategy enables them to benefit from the features of both reproductive modes. In general, asexual reproduction is broadly divided into parthenogenesis and vegetative reproduction. As in parthenogenesis, individuals develop ovaries and lay eggs, the most significant event in switching from parthenogenesis to sexual reproduction is the production of testes. Meanwhile, in vegetative reproduction, individuals do not need germ cells themselves. Thus, they must post-embryonically develop and maintain germ cells derived from pluripotent cells as they switch from vegetative to sexual reproduction. The complicated mechanisms for controlling the postembryonic reproductive development remain unknown. The planarian Dugesia ryukyuensis can switch from vegetative to sexual reproduction by stimulating bioactive compounds called sex-inducing substances, which are widely conserved in Platyhelminthes, including parasitic flatworms. The two reproductive modes are facilitated by the presence of adult pluripotent stem cells, which generate any type of somatic tissue in the asexual state and produce and maintain hermaphroditic reproductive organs in the sexual state. In this study, using RNA sequencing analysis in experimental sexualization by sex-inducing substances, we identified four essential genes for sexualization. A common feature following the knockdown of the four essential genes was a blockage of testicular differentiation. One of the four essential genes was a gap junction gene, Dr-siri (Dugesia ryukyuensis-sexual induction-related innexin). We suggest that the establishment of a testicular stem cell niche supported by Dr-siri protein is responsible for the breakthrough of dormancy in postembryonic reproductive development in planarian reproductive switching. Our findings suggest that the production of testes might be crucial for even switching from vegetative to sexual reproduction.

The spatial and temporal patterns of marine diversity are essential for the sustainable use of fishery resources and the effective management and conservation of marine ecosystems. We investigated the latitudinal diversity gradients of fish genera in Japanese waters using an environmental DNA (eDNA) data. A total of 1,765 environmental DNA surveys across Japan were obtained from the All Nippon eDNA Monitoring Network (ANEMONE) database. We found a negative correlation between fish genus richness and latitude, with a particularly pronounced gradient in summer. Seasonal change in the latitudinal gradient may reflect the increased occurrence of vagrant fish from tropical regions, rather than the commonly assumed pattern of seasonal migration (i.e., southwards in winter and northwards in summer). Furthermore, our results suggests that global warming may lead to a temporary increase in biodiversity by promoting the influx of tropical vagrant fish. However, such increases are unlikely sustainable, as these vagrant species may not establish long-term populations. We refer to this phenomenon as “pseudo biodiversity”, which may give the illusion of increase in biodiversity enhancement in short-term monitoring. This concept warrants attention as similar patterns of apparent biodiversity increase may occur under other circumstances as well.

Periodical cicadas are remarkable for their incredibly long, prime-numbered life cycles and almost perfectly synchronized mass emergence. Synchronized emergence is a generally localized event, referred to as a “brood”. Broods are separated in space and time, or parapatric; adjacent broods emerge on different schedules, whereas some cicadas emerge off schedule, called stragglers. Straggling can potentially erode brood boundaries; thus, the mechanism allowing broods to maintain nonoverlapping distributions is highly puzzling. Here, we propose that predation may allow broods to exclude each other. Our model and numerical simulations show that predation could act as an important factor for maintaining the nonoverlapping distributions. The proposed mechanism is most effective in the vicinity of the critical strength of predation, beyond which the population is doomed to extinction. An increase in predation intensity increases resistance to settlement of a minority brood, while suppressing the main brood population.

In social insect colonies, selfish behaviour due to intracolonial conflict among members can result in colony-level costs despite close relatedness. In certain termite species, queens use asexual reproduction for within-colony queen succession but rely on sexual reproduction for worker and alate production, resulting in multiple half-clones of a single primary queen competing for personal reproduction. Our study demonstrates that competition over asexual queen succession among different clone types leads to the overproduction of parthenogenetic offspring, resulting in the production of dysfunctional parthenogenetic alates. By genotyping the queens of 23 field colonies of Reticulitermes speratus , we found that clone variation in the queen population reduces as colonies develop. Field sampling of alates and primary reproductives of incipient colonies showed that overproduced parthenogenetic offspring develop into alates that have significantly smaller body sizes and much lower survivorship than sexually produced alates. Our results indicate that while the production of earlier and more parthenogenetic eggs is advantageous for winning the competition for personal reproduction, it comes at a great cost to the colony. Thus, this study highlights the evolutionary interplay between individual-level and colony-level selection on parthenogenesis by queens.

A substance that sexualizes planarians, an ancestral group of parasitic flatworms, is widely present in planarians and parasitic flatworms. Here, we present a protocol for extracting and purifying the active fraction with sex-inducing activity. We describe steps for homogenization of flatworms, sample concentration, open-column chromatography, and reverse-phase high-performance liquid chromatography. We then detail a feeding bioassay to confirm sex-inducing activity. The obtained active fraction may positively affect parasitic flatworm sexual maturation and can be tested by adding it into the culture media. For complete details on the use and execution of this protocol, please refer to Sekii et al. (2023).1.

Various parasitic flatworms infect vertebrates for sexual reproduction, often causing devastating diseases in their hosts. Consequently, flatworms are of great socioeconomic and biomedical importance. Although the cessation of parasitic flatworm sexual reproduction is a major target of anti-parasitic drug design, little is known regarding bioactive compounds controlling flatworm sexual maturation. Using the planarian Dugesia ryukyuensis, we observed that sex-inducing substances found in planarians are also widespread in parasitic flatworms, such as monogeneans and flukes (but not in tapeworms). Reverse-phase HPLC analysis revealed the sex-inducing substance(s) eluting around the tryptophan retention time in the fluke Calicophoron calicophorum, consistent with previous studies on the planarian Bipalium nobile, suggesting that the substance(s) is likely conserved among flatworms. Moreover, six of the 18 ovary-inducing substances identified via transcriptome and metabolome analyses are involved in purine metabolism. Our findings provide a basis for understanding and modifying the life cycles of various parasitic flatworms.

Selfish genetic elements (SGEs) increase their transmission efficiency relative to the rest of the individual genome, which is often deleterious to individual fitness. Theoretical studies have suggested that intragenomic conflict over the sex ratio distortion between SGEs and the rest of the genome should lead to the evolution of sex-determining systems. However, in insects, there are relatively few studies other than those on Dipterans, which makes it difficult to understand the role of SGEs in the evolution of insect sex determination. This is partially due to the difficulties in observing SGEs under field conditions. The effect of SGEs is often masked by the counter-evolution of the resistance genes. Interpopulation cross-breeding experiments are effective to detect the SGEs and their resistance genes. If these populations have different SGEs and resistance genes, cross-breeding experiments reveal their existence by collapsing the evolutionary antagonistic state. The Ryukyu drywood termites Neotermes sugioi , distributed in the Ryukyu Islands, show male-biased sex ratios in pseudergates, nymphs, alates and soldiers both in Okinawa and Ishigaki Islands, but different degrees of bias have been reported between the islands. Male-specific microsatellite alleles have been reported in this species, which allowed us to identify the sex of the eggs and young larvae. In this study, we used the microsatellite locus with male-specific alleles to investigate the primary sex ratio of field colonies on Okinawa and Ishigaki islands and the sex ratio of offspring obtained through cross-breeding experiments between the islands. The primary sex ratios of field colonies were male-biased in Okinawa but not in Ishigaki. Cross-breeding experiments showed that Okinawa males tend to have a male-biased sex ratio in their offspring, but Ishigaki males do not. This result is consistent with the hypothesis that the male bias in this species is caused by SGEs, even though termites are phylogenetically distant from Diptera. Accumulation of knowledge on genetic conflicts in a wide range of taxa might be an important step toward elucidating the mechanisms of diversification of sex determination systems in insects.

The origin of eusociality, altruistically foregoing personal reproduction to help others, has been a long-standing paradox ever since Darwin. Most eusocial insects and rodents likely evolved from subsocial precursors, in which older offspring “helpers” contribute to the development of younger siblings without a permanent sterile caste. The driving mechanism for the transition from subsociality (with helpers) to eusociality (with lifelong sterile workers) remains an enigma because individuals in subsocial groups are subject to direct natural selection rather than kin selection. Our genomic imprinting theory demonstrates that natural selection generates eusociality in subsocial groups when parental reproductive capacity is linked to a delay in the sexual development of offspring due to sex-antagonistic action of transgenerational epigenetic marks. Focusing on termites, our theory provides the missing evolutionary link to explain the evolution of eusociality from their subsocial wood-feeding cockroach ancestors, and provides a novel framework for understanding the origin of eusociality.

Sexually mature planarians produce sex-inducing substances that induce postembryonic development of hermaphroditic reproductive organs in asexual freshwater planarians. Although the sex-inducing substances may be useful for elucidating the mechanism underlying this reproductive switch, the available information is limited. The potency of sex-inducing activity is conserved, at least at the order level. Recently, we showed that the sex-inducing activity in the land planarian Bipalium nobile was much higher than that in freshwater planarians. In the present study, we performed bioassay-guided fractionation of the sex-inducing substances produced by B. nobile and propose that crucial sex-inducing activity that triggers complete sexualization for asexual worms of the freshwater planarian Dugesia ryukyuensis is produced by additive and/or synergetic effects of various sex-inducing substances involved in ovarian development. The current study provided an isolation scheme for the minimum-required combination of sex-inducing substances for producing crucial sex-inducing activity.

All animals, other than Platyhelminthes, produce eggs containing yolk, referred to as “entolecithal” eggs. However, only Neoophora, in the phylum Platyhelminthes, produce “ectolecithal” eggs (egg capsules), in which yolk is stored in the vitelline cells surrounding oocytes. Vitelline cells are derived from vitellaria (yolk glands). Vitellaria are important reproductive organs that may be studied to elucidate unique mechanisms that have been evolutionarily conserved within Platyhelminthes. Currently, only limited molecular level information is available on vitellaria. The current study identified major vitellaria-specific proteins in a freshwater planarian, Dugesia ryukyuensis, using peptide mass fingerprinting (PMF) and expression analyses. Amino acid sequence analysis and orthology analysis via OrthoFinder ver.2.3.8 indicated that the identified major vitellaria-specific novel yolk ferritins were conserved in planarians (Tricladida). Because ferritins play an important role in Fe (iron) storage, we examined the metal elements contained in vitellaria and ectolecithal eggs, using non-heme iron histochemistry, elemental analysis based on inductively coupled plasma mass spectrometry and transmission electron microscopy– energy-dispersive X-ray spectroscopy analysis. Interestingly, vitellaria and egg capsules contained large amounts of aluminum (Al), but not Fe. The knockdown of the yolk ferritin genes caused a decrease in the volume of egg capsules, abnormality in juveniles, and increase in Al content in vitellaria. Yolk ferritins of D. ryukyuensis may regulate Al concentration in vitellaria via their pooling function of Al and protect the egg capsule production and normal embryogenesis from Al toxicity.

Background Many animals switch between asexual and sexual reproduction in nature. We previously established a system for the sexual induction of planarian Dugesia ryukyuensis by feeding asexual planarians with minced sexual planarians. We identified dl -tryptophan (Trp) as one of the sex-inducing substances. dl -Trp can induce ovarian development, the first and essential step of sexual induction. d -Trp must act as a principal bioactive compound in terms of ovarian development, because the ovary-inducing activity of d -Trp was 500 times more potent than that of l -Trp. However, how Trp controls sexual induction is still unknown. Results In this study, qRT-PCR analyses suggested that the putative amino acid transporter gene Dr-SLC38A9 is highly expressed in sexual worms, especially in the yolk glands. In situ hybridization analyses showed that Dr-SLC38A9 is expressed in the ovarian primordia of asexual worms and in the mature ovaries, testes, and yolk glands of sexual worms. In addition, Dr-SLC38A9 RNA interference during sexual induction resulted in the suppression of the development of reproductive organs. These results suggest that Dr-SLC38A9 is involved in the development of these organs. Moreover, we demonstrated that the reproductive organ-specific expression of Dr-SLC38A9 is enhanced by the addition of d -Trp. Conclusion We propose that d -Trp activates the expression of Dr-SLC38A9 to promote sexual induction in the planarian D. ryukyuensis .

Sexual dimorphism is a pervasive form of variation within species. Understanding how and why sexual dimorphism evolves would contribute to elucidating the mechanisms underlying the diversification of traits. In flowering plants, pollinators are considered a driver of sexual dimorphism when they affect female and male plant fitness in distinct ways. Here, we found that flowers appear to manipulate the behavior of pollinators using sexually dimorphic traits in the dioecious tree Eurya japonica . In this plant, female flowers present a higher-quality reward for pollinators, whereas male flowers have a more conspicuous appearance. Plants benefit by inducing pollinators to carry pollen from male to female flowers, and their sexual dimorphism might thus facilitate pollen movement through pollinator behavior. In two-choice experiments, pollinators frequently moved from male to female flowers, whereas computer simulation suggested that sexually dimorphic traits would evolve if pollinators changed behavior depending on the traits of the flowers they had just visited. These results suggest that the floral traits affecting the visiting order of pollinators have evolved in plants. Using E. japonica , we theoretically show that the induction of sequential behavior in pollinators might be crucial to the evolution of sexual dimorphism in flowers, and our experiments support these findings.

1. To clarify how intraspecific competition over fertilization affects population growth rates and community dynamics in plants, Kobayashi (Journal of Ecology, 2019) proposed analytical and simulation models based on the game theory. Within this framework, it is suggested that the negative density-dependent effect arising from sexual selection is an important force for sustaining biodiversity. Relaxing certain assumptions made in the analytical model, Iritani (Journal of Ecology, 2019) successfully incorporated the effect of kin selection in the model. This change makes a significant contribution towards improving the generality of the model. 2. Here, I clarify the contribution of Iritani (Journal of Ecology, 2019) and explain the condition under which kin selection promotes the evolution of cooperation (facilitativeness). 3. Kin selection has the potential to promote the evolution of cooperation under certain conditions. However, it is difficult to maintain cooperative traits in high-density environments where individuals must interact with many other conspecifics. Thus, even after the model was extended by Iritani, it cannot be used to state that the negative density-dependent effect arising from intraspecific competition over fertilization does not exist, but it has successfully revealed that the situation is more complex in nature. 4. Synthesis. Kin selection exists wherever intraspecific interactions between individuals occur, but does not necessarily lead to the evolution of cooperation. More detailed theoretical and empirical studies are needed to determine whether sexual selection generally produces negative density dependence, even when kin selection is taken into account.

Evolutionary ecological theory suggests that selection arising from interactions with conspecifics, such as sexual and kin selection, may result in evolution of intraspecific conflicts and evolutionary 'tragedy of the commons'. Here, we propose that such an evolution of conspecific conflicts may affect population dynamics in a way that enhances species coexistence. Empirical evidence and theoretical models suggest that more abundant species is more susceptible to invasion of 'selfish' individuals that increase their own reproductive success at the expense of population growth (intraspecific adaptation load). The density-dependent intraspecific adaptation load gives rise to a self-regulation mechanism at the population level, and stabilizes species coexistence at the community level by negative frequency-dependence.

Synergy is known to be vital for the group collaboration among non-kin individuals. In order to evaluate the condition of synergy that initiates group living, we build a model of food intake based on three types of functional response. We show that type III functional response is prerequisite for synergy to allow group living. The optimal number of gathering individuals can be also evaluated from Type III functional response curve. Type III functional response consists of terms depending linearly and bilinearly on the number of individuals and the bilinear term represents synergy. For a fixed value of the linear coefficient, there are upper and lower boundaries of the bilinear coefficient for synergistic collaboration. The dilution effect can be incorporated into the model through the functional response of the predator. Thus, the functional response of the predator as well as that of the prey contribute to the group living of the prey. Our model shows that group livings can be categorized into three types, namely those due to (1) synergy effect of the own group, (2) dilution effect against predators, and (3) both effects contributing together. The predator's functional response plays a decisive role in the last two types, where the predator response should be of anti-Type III (i.e., Type II).

Sexual reproduction is a common feature of eukaryotes which include dominant members in many biological communities, especially on the hot spots of biodiversity. Given its ubiquity, sex may have an important role to characterize the community structures. In this chapter, we explore the possibility that sex plays an important role to sustain the community complexities. First, we overview several properties of sex and various phenomena caused by it. Then, we demonstrate the effect of sex on the communities by introducing recent studies and discuss future directions.

Sex-determination systems often show remarkable diversity in upstream signals, although downstream genes are broadly conserved. Therefore, the downstream genes have been investigated in various taxa, but the most upstream signals determining sex in insects have been well-described mainly in model organisms, including fruit flies and honey bees, and not in hemimetabolous insects such as termites. Identification of sex-linked genetic markers in termites is important to the survey of primary sex-determination signals. Here, we report male-specific alleles at the microsatellite locus NK12-1 in the Ryukyu drywood termiteNeotermes sugioi(Kalotermitidae). This study provides the third example of a genetic marker linked with sexual phenotype in termites, which is a small but important step to elucidate the evolutionary process of the sex-determination system in termites.

In metazoans, the separation of somatic and germ cells, referred to as the determination of primordial germ cells, occurs via three mechanisms: preformation, epigenesis, and postembryonic germ cell development. Some metazoans occasionally switch between asexual and sexual reproduction based on environmental changes, life cycle phases, or both. In general, asexual animals possess pluripotent stem cells and can regenerate lost body parts by asexual reproduction. Planarians can undergo "degrowth" under starvation, since their body size is homeostatically regulated by "cell turnover" from neoblasts. The regional distribution of d-amino acids is dependent on the expression of amino acid racemases and d-amino acid degrading enzymes. The crucial sex-inducing substances needed to overcome the point-ofno-return in asexual worms of D. ryukyuensis may be contained in worms of Tricladida, but not in those of Polycladida. The crucial sex-inducing substances we have targeted so far are able to activate all necessary endocrine systems to induce postembryonic reproductive development.