Chizue Hiruta

Methyl farnesoate synthesis is necessary for the environmental sex determination in the water flea Daphnia pulex.

Sex-determination systems can be divided into two groups: genotypic sex determination (GSD) and environmental sex determination (ESD). ESD is an adaptive life-history strategy that allows control of sex in response to environmental cues in order to optimize fitness. However, the molecular basis of ESD remains largely unknown. The micro crustacean Daphnia pulex exhibits ESD in response to various external stimuli. Although methyl farnesoate (MF: putative juvenile hormone, JH, in daphnids) has been reported to induce male production in daphnids, the role of MF as a sex-determining factor remains elusive due to the lack of a suitable model system for its study. Here, we establish such a system for ESD studies in D. pulex. The WTN6 strain switches from producing females to producing males in response to the shortened day condition, while the MFP strain only produces females, irrespective of day-length. To clarify whether MF has a novel physiological role as a sex-determining factor in D. pulex, we demonstrate that a MF/JH biosynthesis inhibitor suppressed male production in WTN6 strain reared under the male-inducible condition, shortened day-length. Moreover, we show that juvenile hormone acid O-methyltransferase (JHAMT), a critical enzyme of MF/JH biosynthesis, displays MF-generating activity by catalyzing farnesoic acid. Expression of the JHAMT gene increased significantly just before the MF-sensitive period for male production in the WTN6 strain, but not in the MFP strain, when maintained under male-inducible conditions. These results suggest that MF synthesis regulated by JHAMT is necessary for male offspring production in D. pulex. Our findings provide novel insights into the genetic underpinnings of ESD and they begin to shed light on the physiological function of MF as a male-fate determiner in D. pulex.

Molecular cloning of doublesex genes of four cladocera (water flea) species

BackgroundThe gene doublesex (dsx) is known as a key factor regulating genetic sex determination in many organisms. We previously identified two dsx genes (DapmaDsx1 and DapmaDsx2) from a freshwater branchiopod crustacean, Daphnia magna, which are expressed in males but not in females. D. magna produces males by parthenogenesis in response to environmental cues (environmental sex determination) and we showed that DapmaDsx1 expression during embryonic stages is responsible for the male trait development. The D. magna dsx genes are thought to have arisen by a cladoceran-specific duplication; therefore, to investigate evolutionary conservation of sex specific expression of dsx genes and to further assess their functions in the environmental sex determination, we searched for dsx homologs in four closely related cladoceran species.ResultsWe identified homologs of both dsx genes from, D. pulex, D. galeata, and Ceriodaphnia dubia, yet only a single dsx gene was found from Moina macrocopa. The deduced amino acid sequences of all 9 dsx homologs contained the DM and oligomerization domains, which are characteristic for all arthropod DSX family members. Molecular phylogenetic analysis suggested that the dsx gene duplication likely occurred prior to the divergence of these cladoceran species, because that of the giant tiger prawn Penaeus monodon is rooted ancestrally to both DSX1 and DSX2 of cladocerans. Therefore, this result also suggested that M. macrocopa lost dsx2 gene secondarily. Furthermore, all dsx genes identified in this study showed male-biased expression levels, yet only half of the putative 5’ upstream regulatory elements are preserved in D. magna and D. pulex.ConclusionsThe all dsx genes of five cladoceran species examined had similar amino acid structure containing highly conserved DM and oligomerization domains, and exhibited sexually dimorphic expression patterns, suggesting that these genes may have similar functions for environmental sex determination in cladocerans.

Roles of ecdysteroids for progression of reproductive cycle in the fresh water crustacean Daphnia magna

IntroductionDaphnia magna exhibits a parthenogenetic reproductive cycle linked to a moulting cycle, but regulatory mechanisms of neither moulting nor reproductive cycle are understood in daphnids. Moulting is regulated by ecdysteroids in insects. A previous study showed that a titre of ecdysteroids changed during the reproductive cycle in D. magna; however, no clear correlation among titre, moulting and reproductive cycles has been proved in daphnids. To understand endocrine mechanisms underlying the coordinated reproductive cycle, we analysed the expression of genes coding for enzymes in ecdysteroids synthesis or inactivation pathways, and the effects of 20-hydroxyecdysone (20E) on moulting and ovulation in D. magna.ResultsWe cloned orthologues of neverland (nvd) and shade (shd) in the ecdysteroids synthesis pathway, and Cyp18a1 in the ecdysteroids inactivation pathway previously identified in insects. Gene expression of Cyp18a1 changed conversely with the fluctuation in ecdysteroids titre during the intermoulting period. Tissue-specific expression analysis of nvd showed a prominent expression in the gut. Furthermore, treatment of adult female D. magna with 20E inhibited moulting and/or ovulation.ConclusionsOur cloning and phylogenetic analyses showed that nvd and shd as well as Cyp18a1 are evolutionary conserved in D. magna, suggesting that these genes appeared in arthropods before the radiation of insects. The gene expression analysis during the reproductive cycle indicated that Cyp18a1 possibly regulates the decline of ecdysteroid titre before moulting and ovulation. Furthermore, the expression of nvd in the gut suggested that ecdysone might be synthesised in the gut. Exogenous 20E-treatment resulted in the failure of not only moulting, but also ovulation, suggesting that a low level of ecdysteroids before moulting is required for moulting and ovulation in D. magna.

Identification of the Precise Kairomone-sensitive Period and Histological Characterization of Necktooth Formation in Predator-induced Polyphenism in Daphnia pulex

Many organisms have the ability to alter their development in the presence of predators, leading to predator-induced defenses that reduce vulnerability to predation. In the water flea Daphnia pulex, small protuberances called ‘neckteeth’ form in the dorsal neck region in response to kairomone(s) released by predatory phantom midges (Chaoborus larvae). Although previous studies suggested that kairomone sensitivity begins when chemoreceptors begin to function during embryogenesis, the exact critical period was unknown to date. In this study, we investigated the period of kairomone sensitivity and the process of necktooth formation in D. pulex through extensive treatments with pulses of kairomone(s). First, we described the time course of embryogenesis, which we suggest should be used as the standard in future studies. We found the kairomone-sensitive period to be relatively short, extending from embryonic stage 4 to postembryonic first instar. We observed cell proliferation and changes in cell structure in response to the kairomone treatment, and propose a model for necktooth formation. Preliminary LiCl treatment suggests the Wnt signaling pathway involved in crest formation as a candidate for the molecular mechanism underlying this process. Our study provides basic insight toward understanding the mechanisms underlying adaptive polyphenism in D. pulex.

Targeted gene disruption by use of transcription activator-like effector nuclease (TALEN) in the water flea Daphnia pulex.

BackgroundThe cosmopolitan microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have its complete genome sequenced, an unprecedented ca. 36% of which has no known homologs with any other species. Moreover, D. pulex is ideally suited for experimental manipulation because of its short reproductive cycle, large numbers of offspring, synchronization of oocyte maturation, and other life history characteristics. However, existing gene manipulation techniques are insufficient to accurately define gene functions. Although our previous investigations developed an RNA interference (RNAi) system in D. pulex, the possible time period of functional analysis was limited because the effectiveness of RNAi is transient. Thus, in this study, we developed a genome editing system for D. pulex by first microinjecting transcription activator-like effector nuclease (TALEN) mRNAs into early embryos and then evaluating TALEN activity and mutation phenotypes.ResultsWe assembled a TALEN construct specific to the Distal-less gene (Dll), which is a homeobox transcription factor essential for distal limb development in invertebrates and vertebrates, and evaluated its activity in vitro by single-strand annealing assay. Then, we injected TALEN mRNAs into eggs within 1 hour post-ovulation. Injected embryos presented with defects in the second antenna and altered appendage development, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene.ConclusionsWe succeeded, for the first time in D. pulex, in targeted mutagenesis by use of Platinum TALENs. This genome editing technique makes it possible to conduct reverse genetic analysis in D. pulex, making this species an even more appropriate model organism for environmental, evolutionary, and developmental genomics.

Spindle Assembly and Spatial Distribution of γ-tubulin during Abortive Meiosis and Cleavage Division in the Parthenogenetic Water Flea Daphnia pulex

In most animal species, centrosomes, the main microtubule-organizing centers, usually disintegrate in oocytes during meiosis and are reconstructed from sperm-provided centrioles before the first cleavage division. In parthenogenetic oocytes, however, no sperm-derived centrosome-dependent microtubule nucleation is expected, as fertilization does not occur. The water flea Daphnia pulex undergoes parthenogenesis and sexual reproduction differentially in response to environmental cues. We used immunofluorescence microscopy with anti-&agr;-tubulin and anti-&ggr;-tubulin antibodies to examine spindle formation and the occurrence of centrosomes during parthenogenetic oogenesis and the subsequent cleavage division in D. pulex. The spindle formed in abortive meiosis in parthenogenesis is barrel-shaped and lacks centrosomes, whereas the spindle in the subsequent cleavage division is typically spindle-shaped, with centrosomes. During abortive meiosis, &ggr;-tubulin is localized along the spindle, while in the first cleavage division it is localized only at the spindle poles. Thus, D. pulex should provide a useful comparative model system for elucidating mechanisms of spindle formation and improving our understanding of how evolutionary modification of these mechanisms is involved in the switch from sexual to parthenogenetic reproduction.

Comparative Developmental Staging of Female and Male Water Fleas Daphnia pulex and Daphnia magna During Embryogenesis

The freshwater crustacean genus Daphnia has been used extensively in ecological, developmental and ecotoxicological studies. Daphnids produce only female offspring by parthenogenesis under favorable conditions, but in response to various unfavorable conditions and external stimuli, they produce male offspring. Although we reported that exogenous exposure to juvenile hormones and their analogs can induce male offspring even under female-producing conditions, we recently established a male induction system in the Daphnia pulex WTN6 strain simply by changing day-length. This male and female induction system is suitable for understanding the innate mechanisms of sexual dimorphic development in daphnids. Embryogenesis has been described as a normal plate (developmental staging) in various daphnid species; however, all studies have mainly focused on female development. Here, we describe the developmental staging of both sexes during embryogenesis in two representative daphnids, D. pulex and D. magna, based on microscopic time-course observations. Our findings provide the first detailed insights into male embryogenesis in both species, and contribute to the elucidation of the mechanisms underlying sexual differentiation in daphnids.

How Does the Alteration of Meiosis Evolve to Parthenogenesis?- Case Study in a Water Flea, Daphnia pulex -

Most daphnid species reproduce parthenogenetically as well as sexually, resulting in the production of diploid progenies in both cases. In natural populations, parthenogenesis is the common mode of reproduction, and parthenogenetic offspring are all female. However, in response to certain environmental conditions, such as crowding or seasonal change, male offspring are also produced parthenogenetically, and sexual reproduction occurs (Hebert, 1978). Although they switch between parthenogenesis and sexual reproduction in response to environmental conditions, little is known about the molecular and cytological mechanisms switching and governing each reproductive mode. It can be interpreted that D. pulex develops a reproductive strategy utilizing ‘parthenogenesis’, which has high reproductive power, and ‘sexual reproduction’, which generates genetic diversity, in response to different environments. These theoretical studies have been made on evolutionary mechanism of reproductive modes (Decaestecker et al., 2009); however, practically no study analyzing the evolutionary mechanism while taking the developmental constraints into consideration has so far been conducted. Our understanding of the evolution of reproductive strategy would increase once we precisely clarify the developmental gene programs operating there. We have recently started to develop D. pulex as an experimental model for studying oogenesis and developmental mechanisms during evolution.

The Behavior of Chromosomes During Parthenogenetic Oogenesis in Marmorkrebs Procambarus fallax f. virginalis

Parthenogenetic oogenesis varies among and even within species. Based on cytological mechanisms, it can largely be divided into apomixis (ameiotic parthenogenesis) producing genetically identical progeny, and automixis (meiotic parthenogenesis) producing genetically non-identical progeny. Polyploidy is common in parthenogenetic species, although the association between parthenogenesis and polyploidy throughout evolution is poorly understood. Marmorkrebs, or the marbled crayfish, was first identified as a parthenogenetic decapod and was tentatively named as Procambarus fallax f. virginalis. Previous studies revealed that Marmorkrebs is triploid and produces genetically identical offspring, suggesting that apomixis occurs during parthenogenetic oogenesis. However, the behavior of chromosomes during the process of oogenesis is still not well characterized. In this study, we observed parthenogenetic oogenesis around the time of ovulation in P. fallax f. virginalis by histology and immunohistochemistry. During oogenesis, the chromosomes were separated into two groups and behaved independently from each other, and one complete division corresponding to mitosis (the second meiosis-like division) was observed. This suggests that parthenogenetic oogenesis in Marmorkrebs exhibits gonomery, a phenomenon commonly found in apomictic parthenogenesis in polyploid animals.

Androgenic Gland Implantation Induces Partial Masculinization in Marmorkrebs Procambarus fallax f. virginalis.

The androgenic gland in malacostracan crustacean species produces and secretes androgenic gland hormone, which is responsible for male sexual differentiation, such as the induction and development of male sexual traits, and in turn the suppression of female sexual traits. Marmorkrebs, Procambarus fallax forma virginalis, which was identified as the first parthenogenetic species in decapod crustaceans, produces only female offspring. In this study, in order to reveal whether the Marmorkrebs crayfish is sensitive to androgenic gland hormone, we transplanted an androgenic gland from a related congener, P. clarkii, to P. fallax f. virginalis. In androgenic gland-implanted specimens, partial masculinization was confirmed: the masculinization of several external sexual characteristics (i.e., thickening of the first and second pleopods; formation of reverse spines on the third and fourth pereopods) was detected, whereas that of internal sexual characteristics (e.g., the formation of ovotestes and male gonoducts) was not. Our results imply that P. fallaxf. virginalis still has sensitivity to the androgenic gland hormone and, at least partly, the hormone should be able to induce male characteristics, even in parthenogenetic Marmorkrebs.