Projects

Last update: 2025.2.7

Our research aims to understand the mechanisms of sex differentiation in crustaceans, focusing on larval metamorphosis, left-right asymmetry, and reproductive systems. Lately, it feels like we spend more time raising animals and conducting field surveys than doing lab experiments—but that's all part of the fun! We use a variety of research tools to uncover the fascinating biological phenomena of these organisms.

In organisms like Daphnia, where the sex of offspring is determined by environmental factors, this system is known as environmental sex determination (ESD). Daphnia flexibly adjust both sex determination and reproductive mode in response to changes in external conditions. So, what exactly constitutes an "unfavorable" environment for Daphnia Over more than 100 years of research, various environmental factors have been identified that promote the production of males and resting eggs. These include day length, water temperature, nutrient availability (food supply), population density, and various combinations of these factors. (Illustration by Ayano Katayama)

1-3: Daphnia as a model organism

Daphnia offer numerous advantages as an experimental organism for research, making them an excellent model system in various fields.

• Easy to culture: Since Daphnia are freshwater organisms, maintaining water conditions is simple. They are fed plant plankton such as Chlorella.

• Rapid reproduction of genetically identical individuals: Daphnia reach sexual maturity in about one week and can reproduce asexually, producing 20-40 offspring every three days. Because offspring produced via parthenogenesis are clones of the mother, it is easy to obtain genetically uniform individuals for experiments.

• Easy to observe: Their transparent bodies allow researchers to easily observe internal organs. Additionally, eggs laid in the brood chamber of the mother continue to develop even after being removed, enabling microscopic observation of growth from egg to juvenile.

• Available genomic information: In 2011, Daphnia pulex became the first crustacean to have its entire genome sequenced (Colbourne et al., Science 2011). The Daphnia Genomics Consortium has since been leading research on the comprehensive analysis of DNA, RNA, proteins, metabolites, and gut microbiota, with an increasing number of datasets being registered in public databases.

• Establishment of microinjection techniques for eggs: For non-model organisms like Daphnia, establishing genetic functional analysis techniques has been a major challenge. In 2011, Dr. Yasuhiko Kato from Osaka University developed a method to introduce foreign substances into Daphnia magna embryos using microinjection (Kato et al., Dev Genes Evol 2011). This enabled RNA interference (RNAi) for the first time in Daphnia.
  In 2013, Dr. Chizue Hiruta further refined the microinjection technique for Daphnia pulex (Hiruta et al., BMC Biotech 2013). Today, microinjection is also used for genome editing in Daphnia, expanding the possibilities for genetic modification research (Toyota et al., RNA interference 2016).

1-4: Molecular basis of environmental sex determination in Daphnia (2010–present)

Sex determination mechanisms in organisms can generally be classified into two types: genotypic sex determination (GSD), which depends on chromosomal composition (e.g., XY/XX, ZZ/ZW), and environmental sex determination (ESD), where external environmental factors influence sex determination. While the molecular mechanisms of GSD have been increasingly understood through research on various model organisms, including humans, ESD remains largely unexplored due to the lack of well-established model organisms and experimental systems. One well-known example of ESD is temperature-dependent sex determination (TSD) in reptiles. In all studied crocodilian species, most turtle species, and some lizards, the incubation temperature of eggs determines the sex of the offspring. However, studying TSD in reptiles is challenging due to their long generation times and the difficulty of maintaining them in laboratory conditions. Thus, identifying an ideal model organism was crucial for advancing ESD research. This is where Daphnia became a focus of study. As mentioned earlier, Daphnia reproduce asexually under favorable conditions, producing almost exclusively female offspring. However, when environmental conditions deteriorate, they switch to male production.

In the early 2000s, two independent research groups from Japan and the U.S. simultaneously discovered that exposing Daphnia to juvenile hormone (JH) could induce male production regardless of environmental conditions (Olmstead and LeBlanc, J Exp Zool 2002; Tatarazako et al., Chemosphere 2003). JH is a highly conserved endocrine factor in arthropods, playing a crucial role in regulating molting, metamorphosis, and sexual maturation. Following this discovery, researchers found that the sensitive period for JH-induced male production occurs in the oocyte stage within the maternal ovary. This means that injecting JH into the mother does not affect the sex of the eggs already in the brood chamber, but rather, influences the eggs still in the ovary, which will later be deposited in the brood chamber. These findings led to the hypothesis that, when a mother perceives environmental deterioration, JH levels rise within her body, influencing oocytes in the ovary to develop into males. However, the molecular mechanisms by which environmental signals are translated into JH levels and ultimately regulate male differentiation remain largely unknown. To uncover the molecular basis of ESD in Daphnia, I have been using JH as a key factor in my research.

Related publications & reviews

• Expression analysis of the sex-determination gene doublesex across cladoceran species (Toyota et al., BMC Genomics 2013)

• Isolation and ligand specificity analysis of JH receptors from Daphnia pulex and Daphnia magna (Miyakawa et al., Nature Communications 2013)

• Establishment of a photoperiod-dependent sex induction system using Daphnia pulex WTN6 and discovery of JH biosynthesis involvement in male production (Toyota et al., J Insect Physiol 2015)

• Transcriptome analysis identifies ionotropic glutamate receptors as upstream regulators of JH-mediated male induction in WTN6 (Toyota et al., BMC Genomics 2015)

• Metabolomic analysis identifies pantothenic acid (vitamin B5) as a male-inducing factor in WTN6 (Toyota et al., Sci Rep 2016)

• Developmental stage chart of embryogenesis in Daphnia pulex and Daphnia magna (Toyota et al., Zool Sci 2016)

• Microarray analysis catalogs genes in the ovary that respond to JH during the sex-determination period (Toyota et al., J Appl Toxicol 2016)

• Review on Daphnia microinjection techniques (Toyota et al., InTech 2017)

• Discovery of the involvement of the protein kinase C pathway in WTN6 male induction (Toyota et al., Biol Open 2017)

• Detailed photoperiodic study on male induction in WTN6 (Toyota et al., Zool Sci 2017)

• Comprehensive review of environmental sex determination in Daphnia (Toyota et al., Springer 2018)

• Transgenerational effects of photoperiod exposure on life-history traits in a 50-year-old resurrected Daphnia magna population (Toyota et al., Sci Rep 2019)

• Systematic review of signal pathways involved in male induction in two photoperiod-dependent Daphnia magna strains (Toyota et al., J Appl Toxicol 2020)

• Quantitative analysis of sex-specific swimming behavior in Daphnia magna (Toyota et al., J Exp Zool 2022)

1-5: Chemical safety testing using Daphnia (2010–present)

Daphnia are highly sensitive to environmental changes and water pollution caused by chemicals. Because of this, they have long been used as bioindicators for monitoring environmental pollution in natural ecosystems. Their role extends beyond Japan, as the Organisation for Economic Co-operation and Development (OECD) has included two standardized toxicity tests using Daphnia (mainly Daphnia magna) in its chemical evaluation guidelines (OECD Test Guideline 202; 211).

These tests evaluate:

• Acute toxicity (48-hour survival rate)

• Reproductive toxicity (21-day reproductive rate)

In recent years, toxicology research has focused on understanding how environmental chemicals disrupt intracellular signaling pathways and ultimately reduce survival and reproduction rates. My research specifically investigates how juvenile hormone (JH)-active chemicals affect gene expression in Daphnia. I use microarray analysis and other techniques to examine the molecular mechanisms behind these disruptions.

Related publications & reviews

• Comprehensive screening of JH-responsive genes in Daphnia neonates exposed to three JH-active compounds (fenoxycarb, epofenonane, methoprene) (Toyota et al., J Appl Toxicol 2014)

• Evaluation of JH activity in the chemical compound diofenolan using Daphnia magna (Abe et al., Aqua Toxicol 2015)

• Toxicity assessment of the chemical dispersant Corexit 9500 in Daphnia magna (Toyota et al., J Appl Toxicol 2016)

• Review on the establishment of an Adverse Outcome Pathway (AOP) for the molting hormone in arthropods (You et al., Env Sci Technol 2017)

• Prediction of ligand binding affinity to Daphnia JH receptor genes using docking simulation (Hirano et al., Chem Res Toxicol 2020)

• Review on environmental sex determination in Daphnia and the development of an AOP for juvenile hormone action (Toyota et al., Aqua Toxicol 2022)

2-2: Physiology of sinus gland hormones

Our research focuses on economically important fisheries species in Japan, such as kuruma shrimp Marsupenaeus japonicus and snow crab Chionoecetes opilio. By identifying, isolating, and purifying sinus gland hormones and analyzing their physiological functions, we aim to uncover the roles these hormones play in crustacean biology.

Related publications & reviews

• Comprehensive review on sex determination and differentiation in decapods and cladocerans, focusing on endocrine factors (Toyota et al., Genes 2021)

• Annual reproductive ecology of Leptochela japonica in Ise Bay (Yamane et al., Fish Sci 2022)

• Establishment of an organic synthesis method for androgenic gland hormones in Procambarus clarkii and Procambarus virginalis, successfully inducing testis development in P. virginalis (Katayama et al., Bioorg Chem 2022)

• Isolation and physiological activity analysis of red pigment-concentrating hormone in Pandalus eous (Ohira et al., Aqua Anim 2022)

• Mechanism of calcium deposition in the exoskeleton of Marsupenaeus japonicus (Sekimoto et al., 2022)

• Exploration of luminescence-regulating hormones in Antarctic krill (Ohira et al., Sci J Kanagawa Univ 2022)

• Study on molt-inhibiting hormone in Litopenaeus vannamei (Ohira et al., Sci J Kanagawa Univ 2022)

• Discovery that methyl farnesoate levels increase in the hemolymph of Chionoecetes opilio after the terminal molt (Toyota et al., 2023)

• RNA sequencing of eyestalk ganglia in male and female Marsupenaeus japonicus, isolating two doublesex genes (Toyota et al., Gene 2023)

• Identification of two physiological functions (growth and body color regulation) of crustacean female hormone in juvenile Marsupenaeus japonicus (Toyota et al., Gen Comp Endocrinol 2023)

• Physiological activity analysis of crustacean hyperglycemic hormone in Panulirus japonicus (Toyota et al., Zool Sci 2023)

3. Physiological and ecological studies of invertebrates, focusing on crustaceans (2020–present)

In June 2020, I joined Niigata University’s Sado Marine Biological Station, which marked the beginning of a new research direction—integrating field studies with my previous lab-based work to explore the physiology and ecology of crustaceans. My primary field sites include the Japan Sea region, particularly Sado Island, the Noto Peninsula, and the Oki Islands. Below are some of the key research themes I am currently pursuing. Beyond these topics, I also conduct surveys on other invertebrates that are “reasonably abundant and relatively easy to maintain in captivity”, broadening the scope beyond just crustaceans.

Related publications & reviews

• Plasticity of photoperiod-dependent reproductive modes in Artemia (Toyota et al., Aqua Anim 2021)

• Discovery that juvenile Procambarus clarkii exhibits altered growth rates under blue light conditions (Toyota et al., Zool Stu 2022)

• Optimization of larval diets for Sesarmops intermedium, Metasesarma obesum, and Chiromantes dehaani in aquaculture settings (Toyota et al., Plankton Benthos Res 2023)

• Identification of dominant rhizocephalan parasites and host feminization in Pachygrapsus crassipes  from Manazuru (Kanagawa), Sado (Niigata), and Noto (Ishikawa), Japan (Toyota et al., Zool Sci 2023)

• Symbiotic ecology of Diogenes edwardsii and sea anemones (Toyota & Tsunoda, Japan Sea Research 2024)

4. Sex and lineage identification through morphometric analysis (2019–present)

Inspired by a horsehair crab Erimacrus isenbeckii fisherman’s remark, I embarked on a challenge to distinguish male and female crabs using morphometric data alone. This experience opened my eyes to how much information can be extracted with just a ruler or caliper, leading me to incorporate morphometric analysis into various research projects. This approach not only provides a practical method for species identification and sex differentiation but also offers an opportunity to closely observe and appreciate the intricate details of an organism’s form, deepening our understanding of its functional morphology and evolutionary adaptations.

Related publications & reviews

• Sex differentiation in Erimacrus isenbeckii using geometric morphometric analysis of two-dimensional carapace shape data (Toyota et al., Aqua Anim 2020)

• Establishment of a novel sex differentiation method using three-dimensional carapace morphology in Erimacrus isenbeckii (Toyota et al., Aqua Anim 2021)

• Development of a machine-learning-based sex differentiation method for Erimacrus isenbeckii using carapace images (Ueki et al., Sci Rep 2023)

• Sex and population differentiation in Chiromantes haematocheir based on carapace morphology in Noto (Ishikawa) and Takehara (Hiroshima), Japan (Toyota et al., Japan Sea Research 2024)

• Sex and population differentiation in Ocypode stimpsoni based on carapace morphology in Noto (Ishikawa) and Sado (Niigata), Japan (Toyota et al., Japan Sea Research 2024)

5. Biodiversity and conservation ecology research (2023–present)

During field surveys, I occasionally encounter organisms that are rarely seen—or sometimes entirely new to science. These findings can include:

• Undescribed species that have never been recorded anywhere in the world

• Species discovered outside their known range

• Gynandromorphic individuals (sex mosaics)

• Unusual color or morphological variations

In addition to these discoveries, I believe that documenting long-term observations and collection records from local communities and enthusiasts is crucial for advancing biodiversity research and conservation efforts. Following this philosophy, I have started an initiative to record and archive such findings, collaborating with students, experts, and amateur naturalists. My research extends beyond coastal environments to include deep-sea habitats, beaches, rivers, rice paddies, terrestrial areas, and forests. Additionally, I am expanding my expertise to cover not only invertebrates but also vertebrates and plants, broadening the scope of biodiversity studies.

Related publications & reviews

• Discovery of a Gaetice depressus individual with leucistic left chela (Tsunoda & Toyota, Cancer 2023)

• First record of Mecynotarsus tenuipesfrom Dōgo, Oki Island (Tsunoda et al., Sayabane 2023)

• First record of Quedius japonicus from the Oki Island (Tsunoda et al., Sayabane 2023)

• Stranding record of a fur seal Callorhinus ursinus on Dōgo Island, Oki Archipelago (Tsunoda et al., San’in Natural History Research 2023)

• First record of Carcharhinus brachyurus from Ishikawa Prefecture (Tsunoda et al., Aquatic Animals 2023)

• Intersex Chionoecetes opilio landed in Fukui Prefecture (Tsunoda et al., Aquatic Animals 2023)

• Two cases of morphological abnormalities in Chionoecetes opilio from Fukui Prefecture (Tsunoda et al., Aquatic Animals 2023)

• Discovery of a morphologically variant Kraussia integra in Noto, Ishikawa Prefecture (Tsunoda et al., Noto Marine Center Research Bulletin 2023)

6. Other research topics

During my NNCT years (equivalent to high school + four years of undergraduate studies), I conducted research on molecular markers for selecting valuable persimmon varieties. After earning my Ph.D., I expanded my research beyond crustaceans, collaborating on studies involving various vertebrates, including:

• Reptiles (Mauremys reevesii, Trachemys scripta elegans, Pelodiscus sinensis)

• Mammals (Mus musculus)

• Teleost fish (Oryzias latipes and various marine species)

Related publications & reviews

• Review on functional differentiation of estrogen receptor (ESR) and androgen receptor (AR) (Ogino et al., J Steroid Biochem Mol Biol 2018)

• Identification of GPCR-type ESR in medaka Oryzias latipes (Miyaoku et al., J Appl Toxicol 2021)

• Functional analysis of mesenchymal ESR1 in the mouse uterus (Furuminato et al., Sci Rep 2023)

• Determination of plasma cortisol levels in Girella punctata under high-density rearing in surface vs. deep seawater conditions (Ikari et al., Data Brief 2023)

• Discovery that kynurenine reduces cortisol levels in Paralichthys olivaceus (Ikari et al., Sci Rep 2023)

• Deep-sea water prevents weight loss in Todarodes pacificus by altering cholesterol and mineral metabolism (Hatano et al., Sci Rep 2023)

• Physiological functions of stanniocalcin in goldfish Carassius auratus (Kuroda et al., J Biol Reg Homeo Agents 2023)

6-2: Temperature-dependent sex determination (TSD) in turtles (2018–2023)

Some reptiles exhibit temperature-dependent sex determination (TSD), where the incubation temperature of eggs determines the sex of the offspring. During my postdoctoral research, I participated in a project aiming to uncover the molecular mechanisms of TSD in reptiles. As part of this study, I conducted research using Chinese softshell turtles Pelodiscus sinensis, which exhibit genotypic sex determination (GSD), and Reeves' pond turtles Mauremys reevesii, which rely on TSD. This research contributes to a better understanding of how temperature influences gene expression and sex differentiation in reptiles, shedding light on the evolutionary and ecological significance of TSD mechanisms.