NMDA receptor activation upstream of methyl farnesoate signaling for short day-induced male offspring production in the water flea, Daphnia pulex
© Toyota et al.; licensee BioMed Central. 2016
Received: 26 December 2014
Accepted: 24 February 2015
Published: 14 March 2015
The cladoceran crustacean Daphnia pulex produces female offspring by parthenogenesis under favorable conditions, but in response to various unfavorable external stimuli, it produces male offspring (environmental sex determination: ESD). We recently established an innovative system for ESD studies using D. pulex WTN6 strain, in which the sex of the offspring can be controlled simply by changes in the photoperiod: the long-day and short-day conditions can induce female and male offspring, respectively. Taking advantage of this system, we demonstrated that de novo methyl farnesoate (MF) synthesis is necessary for male offspring production. These results indicate the key role of innate MF signaling as a conductor between external environmental stimuli and the endogenous male developmental pathway. Despite these findings, the molecular mechanisms underlying up- and downstream signaling of MF have not yet been well elucidated in D. pulex.
To elucidate up- and downstream events of MF signaling during sex determination processes, we compared the transcriptomes of daphnids reared under the long-day (female) condition with short-day (male) and MF-treated (male) conditions. We found that genes involved in ionotropic glutamate receptors, known to mediate the vast majority of excitatory neurotransmitting processes in various organisms, were significantly activated in daphnids by the short-day condition but not by MF treatment. Administration of specific agonists and antagonists, especially for the N-methyl-D-aspartic acid (NMDA) receptor, strongly increased or decreased, respectively, the proportion of male-producing mothers. Moreover, we also identified genes responsible for male production (e.g., protein kinase C pathway-related genes). Such genes were generally shared between the short-day reared and MF-treated daphnids.
We identified several candidate genes regulating ESD which strongly suggests that these genes may be essential factors for male offspring production as an upstream regulator of MF signaling in D. pulex. This study provides new insight into the fundamental mechanisms underlying how living organisms alter their phenotypes in response to various external environments.
Sex determination is a fundamental developmental process that contributes to the establishment of sexually dimorphic traits, including the sexual differentiation of gonads, and leads to sex-specific differences in behavior and physiology. Sex determination systems can be divided into two major categories: genotypic sex determination (GSD) and environmental sex determination (ESD) [1-3]. GSD is attributed to the genetic segregation of genes, often residing on sex chromosomes that initially trigger and govern the alteration of sex-specific developmental pathways. In contrast, ESD, which has been repeatedly acquired during animal evolution , is initiated by plural external environmental cues such as temperature, photoperiod and population density, that trigger alternative genetic cascades, resulting in the activation of male or female fate-determining genes [5,6].
The cladoceran crustacean genus Daphnia is a representative organism bearing the ESD system. Under natural favorable environmental conditions, Daphnia produce female offspring by parthenogenesis. However, when an adult female receives unfavorable environmental cues such as low temperature, low food quality, high individual density, or short day-length, Daphnia produce male offspring, thus altering their reproductive mode to sexual reproduction [7-10]. Parthenogenesis allows rapid proliferation during favorable seasons whereas sexual reproduction contributes to an increase in genetic diversity and fitness to deal with changing habitat conditions . Thus, the mechanisms underlying sexual fate determination that depend on external environmental conditions are important for daphnids as these will allow them to fit reproductive strategies appropriately to seasonally changing environments .
Previous studies demonstrated that daphnids administrated with juvenile hormones (JHs) or their analogs induced male offspring even under female-producing conditions [12,13]. In response to parental activation of methyl farnesoate (MF: innate JH in daphnids) signaling, doublesex1 is specifically expressed in the male embryos, and is indispensable for the development of male traits such as testis formation and first antenna elongation . These results suggest that parental MF signaling activated by unfavorable environmental cues affects the developing oocytes during the oocyte maturation stage  and determines their sexual fate [14-19]. However, the regulatory mechanisms of MF signaling and the following downstream pathway for male offspring production have not been clarified yet.
We have successfully established an innovative experimental system using D. pulex WTN6 strain. In this strain, the offspring sex can be controlled by simply changing the day length conditions; a mother produces female progeny reared under long-day conditions (14 h light: 10 h dark), whereas male progeny emerges under short-day conditions (10 h light: 14 h dark) .
In this study, to investigate the up- and downstream events of MF signaling, we reared adult D. pulex WTN6 strain under the following conditions: long-day (female-inducing), short-day (male-inducing) and long-day with MF treatment (male-inducing). The gene expression profiles of the ovary and whole body of these adults at the MF-sensitive period for male offspring production were compared by RNA-seq analysis. We found that the expression levels of ionotropic glutamate receptor-related genes had changed significantly in response to the short-day condition, but not to MF treatment. Using pharmacological manipulation of ionotropic glutamate receptors, we demonstrated that N-methyl-D-aspartic acid (NMDA) receptors (a type of ionotropic glutamate receptor) are essential factors for male offspring production in D. pulex acting as an upstream regulator of MF signaling. Our findings not only provide a molecular component to explain the ESD mechanism but also contribute to elucidate how organisms convert environmental information into phenotypic changes.
Results and discussion
Differentially expressed genes in response to short-day and MF treatment
Illumina HiSeq2500 sequencing yielded a total of 530,174,848 paired-end reads (265,087,424 read pairs). The transcriptome assembly process produced 70,229 putative transcripts using Trinity. The N50 value and the mean length of assembled contigs, which are representative statistics of transcriptome assembly, are 3,043 bp and 1,591 bp, respectively. These scores are consistent with recent studies of some insect and crustacean species [21-23], suggesting that our transcriptome data provides a good resource for investigating the molecular mechanisms of ESD in D. pulex. We identified 55,466 ORFs (N50: 1,488 bp; mean length: 856 bp) in the assembled transcript sequences. Among them, 21,191 had significant BLAST similarity hits with publicly available protein sequences, 7,860 were assigned GO terms according to the genome project in D. pulex , and 17,185 were consistent with gene models constructed by the Daphnia Genomics Consortium .
Upstream factors of MF signaling
In this experimental design, we first compared the female-producing long-day condition, male-producing short-day condition and male-producing MF-treated condition. Genes differentially expressed exclusively in the short-day condition were designated as candidates for upstream of MF signaling, whereas mutual genes differentially expressed in both the short-day and MF-treated conditions were designated as candidates for downstream of MF signaling (Figure 1A). Based on these criteria, we identified 16 and 33 DEGs responding to the short-day condition in the ovary and whole body, respectively, as the candidate transcripts regulating the upstream process of MF signaling (Figure 2A, D). In response to short-day (male-producing) condition, four transcripts (e.g., E3 ubiquitin ligase) were more abundant in ovary with log2-transformed fold change (FC) values between 2.42 and 11.19, whereas nineteen transcripts (e.g., rho-associated coiled-coil containing protein kinase, cytochrome P450 CYP4/19/26 subfamilies, and ER-Golgi vesicle-tethering protein p115) were differentially expressed in whole body with log2-transformed FC values between 2.31 and 9.50 (Additional files 3 and 4). However, not only approximately 80% of the candidate genes in both sample categories could be classified into functionally unknown groups (Figure 2B, E), but also molecular functions of these genes annotated with the regulation of the MF signaling remain largely unclear. Then, we next performed gene ontology (GO) enrichment analysis  to provide an overview of the potential candidate gene sets involved in the upstream of MF signaling governing male offspring production in D. pulex.
List of GO terms in the molecular function analyzed by GO enrichment analysis (Extracted from Additional file 5 )
False discovery rate
Signaling receptor activity
Transmembrane signaling receptor activity
Glutamate receptor activity
Ionotropic glutamate receptor activity
Passive transmembrane transporter activity
Gated channel activity
Ligand-gated channel activity
Ligand-gated ion channel activity
Extracellular ligand-gated ion channel activity
Excitatory extracellular ligand-gated ion channel activity
Extracellular-glutamate-gated ion channel activity
Substrate-specific channel activity
Ion channel activity
Cation channel activity
Calcium channel activity
Molecular transducer activity
Signal transducer activity
Intriguingly, intracellular calcium signaling might be activated in response to the short-day condition, because the expression levels of genes associated with intracellular calcium influx, such as calcium channel activity, changed significantly in both the ovary and whole body categories (Table 1, Additional file 5). Previous studies in several insects reported that an elevation of free intracellular calcium modulated by ionotropic glutamate receptors is necessary for increasing JH biosynthesis in the corpora allata, which is a JH-synthesizing organ in insects [26,27]. Therefore, ionotropic glutamate receptors might regulate the intracellular calcium concentration to modulate endogenous MF levels in daphnids as well as in insect species.
List of GO terms in the biological process analyzed by GO enrichment analysis
False discovery rate
Cell surface receptor signaling pathway****
Neurological system process
Amino sugar metabolic process
Glucosamine-containing compound metabolic process
Aminoglycan metabolic process
Chitin metabolic process
Inorganic anion transport
Phosphate ion transport
We also found that sulfotransferase activity and its upper-hierarchy terms (transferase activity, transferring sulfur-containing groups and fucosyltransferase activity, and galactosyltransferase activity), which are terms that belong to the molecular function category, varied significantly only in the ovary in response to the short-day condition (Additional file 4). Although sulfotransferase-related genes might be one of the candidates for the upstream element of MF signaling, a causal relationship between those genes and the regulatory mechanism of MF remains largely unknown. Further analyses will be required to elucidate the molecular functions of sulfotransferase-related genes in the regulation of MF signaling for the ESD system in D. pulex. These findings provide important clues about the molecular signaling cascade regulating male offspring production in response to the short-day condition in D. pulex.
Administration of agonists and antagonists of ionotropic glutamate receptor subtypes
The present results suggest that NMDA receptors act on the upstream of MF signaling, however, signal cascades connecting NMDA receptors and the activation of MF signaling remain largely unknown. Previously, the TGFβ signaling pathway was identified as a potential candidate connecting NMDA receptor to JH synthesis in Drosophila melanogaster . In the corpora allata of D. melanogaster, TGFβ signaling, which is mediated by decapentaplegic (a TGFβ ligand), thickveins and Mothers against decapentaplegic (main components of its pathway), contributes to the regulation of JH biosynthesis via induction of juvenile hormone O-methyltransferase (JHAMT), a critical enzyme of JH synthesis . Our previous study revealed that JHAMT is a key factor for modulating the innate MF levels governing the ESD in D. pulex . Although the expression of TGFβ signaling pathway-related genes did not change between the short-day and the long-day conditions in our RNA-seq experiments, further investigations concerning TGFβ signaling are necessary to elucidate the signal cascades between NMDA receptors and activation of JHAMT expression in D. pulex.
Most aphid species are known to exhibit cyclical parthenogenesis and ESD in a manner much like the daphnids. It has been reported that the autumnal shortened day-length induce the sexual morph that produces male and oviparous female , and topical application of JH to oviparous producer induces the parthenogenetic female in pea aphid Acyrthosiphon pisum . Moreover, recent progress in omics technologies (e.g., genomics, transcriptomics and proteomics) have allowed the large-scale screening of candidate factors responsible for the switch from parthenogenetic morph to sexual morph induced by shortening of the photoperiod [31-35]. Interestingly, juvenile hormone esterase (JHE, JH degradation enzyme) was identified as a key element for the induction of sexual morph by the JH III titer decrease in response to short-day condition . This finding indicates relationship between endogenous JH III/MF titer in the mothers and sexual outcome of the offspring is an opposite phenomenon in pea aphids and daphnids: high innate JH III titer induces female progeny in pea aphids and male progeny in daphnids. NMDA receptor might act in the singling pathway between receptions of shortened day-length and regulation of innate JH III titer in pea aphids as well as in daphnids. To investigate the common principle of ESD system among them, further comparative analyses will be necessary.
Downstream factors of MF signaling
Finally, we screened the downstream candidates of MF signaling as the mutual DEGs in response to both the short-day condition and MF treatment (Figure 1A). We obtained 21 and 102 DEGs in the ovary and whole body, respectively (Figure 2A, D). In response to short-day condition, seventeen transcripts (e.g., drebrins and related actin binding proteins) were more enriched in ovary with log2-transformed FC values between 2.46 and 8.76, whereas twenty-five transcripts (e.g., low-density lipoprotein receptors containing Ca2+-binding EGF-like domains) were differentially expressed in whole body with log2-transformed FC values between 2.11 and 9.06 (Additional files 3 and 4). Further, candidate genes in the whole body contained several serine protease and hemoglobin-related genes, known as MF-responsive genes in daphnids [36,37], implying that this experimental design possesses higher reliability to select factors involved in downstream of MF signaling. In addition, more than 50% of the candidate genes could not be attributed to any annotations (Figure 2C, F), suggesting that those genes might be novel candidates for sexual fate determination via MF signaling in D. pulex (Additional files 8 and 9).
GO enrichment analysis showed that expression levels of genes associated with protein tyrosine kinase activity and calcium ion transmembrane transporter activity terms varied significantly in response to the short-day condition and MF treatment, especially in the ovary (Additional file 5). Although recent studies indicated that JH acts via intracellular-type receptors to modulate downstream gene expression [38-43], some studies implied that JH actions are mediated via plasma membrane-type receptors involving calcium ion and protein kinase C in D. melanogaster  and two crustaceans, barnacle Balanus amphitrite  and the crayfish Cherax quadricarinatus . Based on this knowledge, it is suggested that MF signal transduction from the mother (ovarian tissues) to oocytes is regulated by not only transcriptional gene cascades via intracellular-type JH receptors but also by phosphorylation cascades through the protein kinase C family in the ovary of daphnids. To prove this hypothesis, further exposure experiments using activators and inhibitors of protein kinase C will be required.
Female- and male-inducing conditions in Daphnia pulex strain
The D. pulex WTN6 strain was obtained from the Center for Genomics and Bioinformatics (Indiana University, IN, USA). This strain was maintained in dechlorinated freshwater, which was aerated and filtered through activated carbon for 2 weeks, at 18°C. A 0.04-ml suspension of 4.3 × 108 cells ml−1 of chlorella (Chlorella vulgaris) was added daily to each culture (40 individuals/2 L). To induce male offspring by exogenous administration of methyl farnesoate (MF, Echelon Bioscience, Salt Lake City, UT, USA), we prepared a stock solution of 1 mg/ml MF dissolved in dimethylformamide (DMF; analytical grade, Wako, Osaka, Japan) and kept it at −20°C until use. This stock solution was added directly to each 50 ml of breeding water (final concentration: 0.8 μM) containing one adult female at 30 h after ovulation (Figure 1A). We confirmed the sex of offspring based on the length of the first antenna  using a Leica MZ FLIII microscope (Leica, Mannheim, Germany).
Chemicals and treatment procedures
We used three agonists of ionotropic glutamate receptors; N-methyl-D-asparatic acid (NMDA) (≥98%; Sigma-Aldrich, St. Louis, MO), (±)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), (≥98%; Sigma-Aldrich), and kainic acid n-hydrate (Kainate) (≥98%; Wako), and two antagonists; (+)-MK-801 hydrogen maleate (MK-801) (≥98%; Sigma-Aldrich) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt hydrate (NBQX) (≥98%; Sigma-Aldrich). Chemicals dissolved in water were stored as 10 mM stock solutions, and kept at 4°C until use. This stock solution was added to each 5 ml of breeding water containing one adult female (one-month old or older) at 30 h after ovulation in a 5-ml sampling tube (INA OPTICA, Osaka, Japan). A total of 15 individuals were used for these experiments. The concentrations of MF, agonists and antagonists used are as follows: MF (0.8 μM), MK-801 (20 μM), NBQX (100 and 200 μM), NMDA, AMPA and Kainate (100 μM). Differences between treatments were statistically analyzed by Fisher’s exact probability test with Holm’s correction using R 2.15.3 .
RNA extraction and sequencing
One individual was cultured in 50 ml of rearing water under the long-day, short-day conditions and long-day condition with MF treatment. They were sacrificed when one month old (i.e., at least 8 times ovulated) during the MF-sensitive period for sex determination of the embryos (50 h after ovulation, Figure 1A). Whole body samples with developing embryos removed from the dorsal chamber and ovary samples consisted of three individuals/replicate, and triplicates were prepared for each experimental condition (long-day, short-day and MF-treated conditions), using a total of 54 individuals. Total RNA was extracted using the RNAqueous-Micro kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The RNA treated with RNase-free DNase was cleaned up using the RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The quality and concentration of total RNA was validated by NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA), Qubit (Life Technologies), and 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The samples for transcriptome analyses were prepared from 1.0 μg of total RNA using TruSeq RNA Sample Preparation v2 kit (Illumina, San Diego, CA, USA) following the manufacturer’s protocols with minor modifications: RNA fragmentation was conducted for 4 min instead of 8 min at 94°C and the number of PCR cycles was reduced to 6. We validated the cDNA libraries using the Bioanalyzer High Sensitivity DNA Assay (Agilent Technologies) and KAPA Library Quantification kits (Kapa Biosystems, Woburn, MA USA) according to the manufacturers’ protocols. Multiplex sequencing of 101 bp paired-end reads was performed on an Illumina HiSeq2500 instrument. The output sequence quality was inspected using the FastQC program . The RNA-Seq reads are available through DRA under the accession number DRA002725.
RNA-seq de novo assembly and annotation
The reads were cleaned up with cutadapt , trimming low-quality ends (< QV30) and adapter sequences, and reads shorter than 50 bp were discarded. Cleaned reads from all libraries were assembled together using the RNA-seq de novo assembler Trinity  in the paired-end mode with the options ‘–min_kmer_cov = 2, −−dnorm_max_cov = 100’. ORFs larger than 150 bp were extracted from the Trinity contigs using TransDecorder, which is included in the Trinity suite. The translated protein sequences were subjected to similarity searches against NCBI nr using the BLASTP program and assigned the functional annotations of the most similar protein sequences. In most cases, the top hits were D. pulex proteins deposited in the RefSeq database. Gene model and annotation were assigned to each constructed transcripts according to D. pulex genome project data .
Differential expression analysis
To identify differentially expressed sequences, we first mapped the reads back to the contigs assembled by Trinity using Bowtie 2 version 2.1.0 . For read mapping, we used a reporting option “-a” in Bowtie 2. Then transcript abundance was estimated by using eXpress version 1.5.1 . We used the edgeR package  of Bioconductor to identify genes that are differentially expressed between each condition following the developer’s manual (false discovery rate: FDR < 0.05). To adjust for library sizes and skewed expression of transcripts, the estimated abundance values were normalized using the Trimmed Mean of M-values (TMM) normalization method included in the edgeR package . Based on a negative binomial model implemented in edgeR, DEGs among the long-day, short-day and MF-treated conditions were selected in the whole body and ovary, separately.
Gene ontology enrichment analysis
GO terms were assigned to each gene model according to the genome project in D. pulex . GO enrichment analysis was carried out using the gene score resampling method in ErmineJ (v3.0.2) , with full resampling of fold change used as gene scores. Among 7,860 constructed transcripts (total 70,229 transcripts) bearing at least one GO term, GO subsets containing between 5 and 150 genes were used in this analysis, and GO terms with the Benjamini-Hochberg FDR < 0.1 were considered as significant . QuickGO was used to provide co-occurrence GO terms which are most often annotated to the same proteins as the selected term .
We would like to thank Drs. John K. Colbourne, University of Birmingham, Taro Maeda, Kota Ogawa and Eiji Watanabe, National Institute for Basic Biology (NIBB), for their technical advice and materials; Ms. Sachiko Wakazuki and Miwako Matsumoto, NIBB, for their technical support for NGS sequencing; members of the Iguchi laboratory for helpful advice and constructive criticism. D. pulex genomic sequence data was produced by The Center for Genomics and Bioinformatics at Indiana University and distributed via wFleaBase in collaboration with the Daphnia Genomics Consortium (https://wiki.cgb.indiana.edu/display/DGC/Home). This work was supported by a JSPS Research Fellowship for Young Scientists (KT) (No.12 J05579), a Sasakawa Scientific Research Grant from The Japan Science Society (KT), a Saito Ho-on Kai Scientific Research Grant from The Saito Gratitude Foundation (KT), grants from the Ministry of Education, Culture, Sports, Science and Technology (YO, SM, TI), the Ministry of the Environment of Japan (NT, TI), a grant from the National Institute for Basic Biology (TI), and the Research Council of Norway (project 221455), and Adverse Outcome Pathways for Endocrine Disruption in Daphnia magna, a conceptual approach for mechanistically-based risk assessment (TI).
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