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Mechanisms underlying sex determination and differentiation in animals are known to encompass a diverse array of molecular clues. Recent innovations in high-throughput sequencing and mass spectrometry technologies have been widely applied in non-model organisms without reference genomes. Crustaceans are no exception. They are particularly diverse among the Arthropoda and contain a wide variety of commercially important fishery species such as shrimps, lobsters and crabs (Order Decapoda), and keystone species of aquatic ecosystems such as water fleas (Order Branchiopoda). In terms of decapod sex determination and differentiation, previous approaches have attempted to elucidate their molecular components, to establish mono-sex breeding technology. Here, we overview reports describing the physiological functions of sex hormones regulating masculinization and feminization, and gene discovery by transcriptomics in decapod species. Moreover, this review summarizes the recent progresses of studies on the juvenile hormone-driven sex determination system of the branchiopod genus Daphnia, and then compares sex determination and endocrine systems between decapods and branchiopods. This review provides not only substantial insights for aquaculture research, but also the opportunity to re-organize the current and future trends of this field.

Background Animals in diverse aquatic groups construct tubes using mucus and filaments, and the acquisition of this capability has likely played an important role in the evolution and diversification of small benthic animals. Tanaidacea is a crustacean order that includes tube-constructing species, most of which belong to Tanaidoidea and Paratanaoidea, with a few in Kalliapseudidae (Apseudoidea). Two previously reported systems used in tube construction are the thoracic-gland system, with secretory glands in thoracic segments (pereonites), and the pereopodal-gland system, with glands in pereopods. Results Parapseudidae (Apseudoidea) also includes a tube-constructing species, Parapseudes algicola (Shiino, 1952), which lacks large secretory glands in all pereonites and pereopods, but has a pair of acinar glands in the pleotelson, lateral to the gut. Each gland connects to the gut via a short duct, and thence to the exterior via the anal opening. Secretions released from these glands are used to construct tubes, and contain acidic and neutral mucopolysaccharides. Conclusion We report in P. algicola a third, novel secretory system, here termed the pleotelsonal-gland system, used for tube construction in Tanaidacea. It is similar to the secretory system in some “thalassinidean” decapods; both systems have secretory glands connecting to the gut and thence to the anal opening as the outlet; however, these gland systems likely evolved independently. Recent discoveries of novel secretory systems for tube construction in Tanaidacea suggest that information from smaller, less well-known groups will be necessary to understand how acquisitions of tube-constructing capability affected diversification in animals.

Background The 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. Results We 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 . Conclusions The 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.

Background The ubiquitous, freshwater microcrustacean Daphnia pulex provides a model system for both human health research and monitoring ecosystem integrity. It is the first crustacean to have a well annotated, reference genome assembly that revealed an unusually high gene count highlighted by a large gene orphanage,-i.e., previously uncharacterized genes. Daphnia are capable of either clonal or sexual reproduction, making them ideally suited for genetic manipulation, but the establishment of gene manipulation techniques is needed to accurately define gene functions. Although previous investigations developed an RNA interference (RNAi) system for one congener D. magna , these methods are not appropriate for D. pulex because of the smaller size of their early embryos. In these studies, we develop RNAi techniques for D. pulex by first determining the optimum culture conditions of their isolated embryos and then applying these conditions to the development of microinjection techniques and proof-of-principle RNAi experiments. Results We found that isolated embryos were best cultured on a 2% agar plate bathed in 60 mM sucrose dissolved in M4 media, providing optimal conditions for microinjections. Then, we injected double-stranded (ds)RNA specific to the Distal-less gene ( Dll ), which is a homeobox transcription factor essential for limb development in invertebrates and vertebrates. Injected embryos presented with defects in the second antenna and appendage development, and dsRNA induced the degradation of Dll mRNAs, indicating that this technique successfully inhibited transcription of the target gene. Conclusions We developed a microinjection system for RNAi studies in D. pulex . These techniques add to the growing genomic toolbox and enhance the genetic tractability of this important model for environmental, evolutionary, and developmental genomics.

Most daphnid species adopt parthenogenesis and sexual reproduction differentially in response to varied environmental cues, resulting in the production of diploid progenies in both cases. Previous studies have reportedly suggested that daphnids produce their parthenogenetic eggs via apomixis; the nuclear division of mature oocytes should be an equational division similar to somatic mitosis. However, it seems premature to conclude that this has been unequivocally established in any daphnids. Therefore, the objective of our research was to precisely reveal the process and mechanism of parthenogenetic oogenesis and maintenance of diploidy in Daphnia pulex through histology, karyology, and immunohistochemistry. We found that, when a parthenogenetic egg entered the first meiosis, division was arrested in the early first anaphase. Then, two half-bivalents, which were dismembered from each bivalent, moved back to the equatorial plate and assembled to form a diploid equatorial plate. Finally, the sister chromatids were separated and moved to opposite poles in the same manner as the second meiotic division followed by the extrusion of one extremely small daughter cell (resembling a polar body). These results suggest that parthenogenetic D. pulex do not adopt typical apomixis. We hypothesize that D. pulex switches reproductive mode depending on whether the egg is fertilized or not.

With more than 40,000 species, Malacostraca is the most diverse crustacean class. Most malacostracans are gonochoristic, but simultaneous hermaphrodites are also known. Tanaidacea is one of two malacostracan orders that includes simultaneously hermaphroditic species; so far, simultaneous hermaphroditism has been confirmed externally and internally in only two tanaidacean species, both in the genus Apseudes (Apseudidae). Here we show, through external and internal morphological observations of fixed specimens, that the apseudid Falsapseudes bowmani is a simultaneous hermaphrodite, making Falsapseudes the second tanaidacean genus in which simultaneous hermaphroditism has been confirmed both externally and internally. In this species, the epistome (a projection on the clypeus) was thick and elongate in large specimens but was thin and spiniform in smaller specimens; the brooding of eggs or embryos was observed only in thin-epistome individuals, although a pair of ovaries was confirmed in both thick- and thin-epistome individuals. This suggests that individuals with a thick epistome may act as males while also retaining the female reproductive organs.

INTRODUCTION: Daphnia 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. RESULTS: We 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. CONCLUSIONS: Our 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.

Background The 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. Results We 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. Conclusions We 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.