Identification and Physiological Assays of Crustacean Hyperglycemic Hormones in the Japanese Spiny Lobster, Panulirus japonicus.

The Japanese spiny lobster Panulirus japonicus lives on rocky shores and is mainly distributed along the Pacific coast around Japan. Due to the high demand for it, the development of aquaculture systems and increasing its resource volume requires further expansive production. However, a major factor preventing the establishment of aquaculture technology for this lobster is the difficulty with rearing processes from larval to juvenile production. A recent study shed light on the molecular mechanisms underlying larval development from the perspective of physiological functions of endocrine factors such as molting hormones. However, physiological studies of P. japonicus are still lacking. In decapod crustaceans, the X-organ/sinus gland complex is a well-known endocrine system that secretes the crustacean hyperglycemic hormone (CHH)-superfamily peptides that regulate growth, molting, sexual maturation, reproduction, and change in body color. In this study, we identified two CHHs from the sinus glands of P. japonicus using reversed-phase high-performance liquid chromatography in order to elucidate their physiological function for the first time.

Gender identification of the horsehair crab, Erimacrus isenbeckii (Brandt, 1848), by image recognition with a deep neural network.

Appearance-based gender identification of the horsehair crab [Erimacrus isenbeckii (Brandt, 1848)] is important for preventing indiscriminate fishing of female crabs. Although their gender is easily identified by visual observation of their abdomen because of a difference in the forms of their sex organs, most of the crabs settle with their shell side upward when placed on a floor, making visual gender identification difficult. Our objective is to use deep learning to identify the gender of the horsehair crab on the basis of images of their shell and abdomen sides. Deep learning was applied to a photograph of 60 males and 60 females captured in Funka Bay, Southern Hokkaido, Japan. The deep learning algorithms used the AlexNet, VGG-16, and ResNet-50 convolutional neural networks. The VGG-16 network achieved high accuracy. Heatmaps were enhanced near the forms of the sex organs in the abdomen side (F-1 measure: 98%). The bottom of the shell was enhanced in the heatmap of a male; by contrast, the upper part of the shell was enhanced in the heatmap of a female (F-1 measure: 95%). The image recognition of the shell side based on a deep learning algorithm enabled more precise gender identification than could be achieved by human-eye inspection.

Sacculina-Induced Morphological Feminization in the Grapsid Crab Pachygrapsus crassipes.

Rhizocephalan barnacles (Thecostraca: Cirripedia) are parasitic crustaceans that lack appendages, segmentation, and a digestive system in adults, while instead infiltrating their hosts with a nutrient-absorbing system of rootlets. Sacculinids, belonging to the Rhizocephala order, are known for their various parasitization-induced effects on their decapod hosts, such as parasitic castration, reduction in the growth of secondary sexual characteristics, feminization of male crabs, and alteration of host behavior. In this study, we conducted field surveys in Japan at Manazuru Town (Kanagawa) on the Pacific coast, and on Sado Island and Noto Peninsula on the Sea of Japan side, and found that sacculinid-parasite-ratios on the grapsid crab Pachygrapsus crassipes were particularly high on the Sea of Japan coast. Molecular phylogenetic analysis revealed that the Manazuru population forms a single clade with Sacculina yatsui, and both Sado and Noto populations form a single clade with S. confragosa. We further demonstrated that external morphologies of male P. crassipes parasitized by sacculinids were changed to female phenotypes. This host-parasite interaction will be a useful model for understanding molecular mechanisms underlying rhizocephalan-driven morphological and behavioral feminization and castration.

Dual roles of crustacean female sex hormone during juvenile stage in the kuruma prawn Marsupenaeus japonicus.

The crustacean female sex hormone (CFSH) has been identified as a female-specific hormone that plays a crucial role in female phenotype developments in the blue crab Callinectes sapidus. To date, its homologous genes have been reported in various decapod species. Additionally, unlike the blue crab, several species have two different CFSH subtypes. The kuruma prawn Marsupenaeus japonicus is a representative example species of this phenomenon, having two CFSH subtypes identified from the eyestalk (MajCFSH) and ovary (MajCFSH-ov). Eyestalk-type MajCFSH is expressed predominantly in the eyestalk at the same level in both sexes, indicating no female-specificity. Here, we conducted gene knockdown analysis of eyestalk-type MajCFSH using sexually immature juveniles of kuruma prawn (average body length: ∼10 mm) to elucidate its physiological functions. As a result, MajCFSH-knockdown did not affect the development of sex-specific characteristics such as external reproductive organs, while it induced apparent growth suppression in male juveniles, implying that MajCFSH may play a male-biased juvenile growth role. Moreover, MajCFSH-knockdown female and male juveniles changed their body color to become brighter, indicating that MajCFSH has the ability to change body color by dispersing the pigment granules in the chromatophore. Overall, our present study improved our understanding of the physiological roles of CFSH using kuruma prawn.

Sexual dimorphic eyestalk transcriptome of kuruma prawn Marsupenaeus japonicus.

Kuruma prawn (Marsupenaeus japonicus) is a benthic decapod crustacean that is widely distributed in the Indo-West Pacific region. It is one of the most important fishery resources in Japan, but its annual catches have declined sharply since the 1990s. To increase stocks, various approaches such as seed production and aquaculture were attempted. Since the demand for important fishery species, including kuruma prawn, is expected to increase worldwide in the future, there is a need to develop new technologies that will make aquaculture more efficient. Historically, the eyestalk endocrine organ is known to consist of the X-organ and sinus gland (XO/SG) complex that synthesizes and secrets various neuropeptide hormones that regulate growth, molt, sexual maturation, reproduction, and changes in body color. In the current study, eyestalk-derived neuropeptides were identified in the transcriptome. In addition, most orthologs of sex-determination genes were expressed in eyestalks. We identified two doublesex genes (MjapDsx1 and MjapDsx2) and found that MjapDsx1 showed male-biased expression in the eyestalk ganglion with no sex-specific splicing, unlike insect species. Therefore, this study will provide an opportunity to advance the research of neuropeptides and sex determination in the kuruma prawn.

Eyestalk transcriptome and methyl farnesoate titers provide insight into the physiological changes in the male snow crab, Chionoecetes opilio, after its terminal molt.

The snow crab, Chionoecetes opilio, is a giant deep-sea brachyuran. While several decapod crustaceans generally continue to molt and grow throughout their lifetime, the snow crab has a fixed number of molts. Adolescent males continue to molt proportionately to their previous size until the terminal molt at which time an allometric increase in chela size occurs and an alteration of behavioral activities occurs, ensuring breeding success. In this study, we investigated the circulating concentrations of methyl farnesoate (an innate juvenile hormone in decapods) (MF) before or after the terminal molt in males. We then conducted eyestalk RNAseq to obtain molecular insight into the regulation of physiological changes after the terminal molt. Our analyses revealed an increase in MF titers after the terminal molt. This MF surge may be caused by suppression of the genes that encode MF-degrading enzymes and mandibular organ-inhibiting hormone that negatively regulates MF biosynthesis. Moreover, our data suggests that behavioral changes after the terminal molt may be driven by the activation of biogenic amine-related pathways. These results are important not only for elucidating the physiological functions of MFs in decapod crustaceans, which are still largely unknown, but also for understanding the reproductive biology of the snow crab.

Effect of Blue Light on the Growth of the Red Swamp Crayfish Procambraus clarkii Larvae -Seasonal and Sexual Differences.

Organisms have the ability to adapt their behavior and physiology in response to seasonal changes in their habitat's environments. Although it is known that a specific light wavelength affects growth and reproduction in various animal taxa, its effect on sexual and seasonal differences in year-round breeding animals remains unclear. Here, we demonstrate that a blue light stimulus promotes or suppresses larval growth in the red swamp crayfish Procambarus clarkia depending on the season. During the spawning season (natural growing period), blue light irradiation accelerates female growth faster than in males, but suppresses growth in both females and males in the overwintering season. Moreover, these seasonal plastic effects of blue light show apparent sexual differences, with female juveniles exhibiting the greatest sensitivity. Our findings provide an opportunity to research how the red swamp crayfish can adapt to various habitable niches from the point of view of light color perception, and can be applied for the development of a more effective aquaculture system, not only for crayfish, but also for other commercially available decapod crustaceans using a specific light environment.

Chemical synthesis and functional evaluation of the crayfish insulin-like androgenic gland factor.

Insulin-like androgenic gland factor (IAG) from the marbled crayfish Procambarus virginalis is an insulin-like heterodimeric peptide composed of A and B chains and has an Asn-linked glycan at the B chain. IAG is considered to be a male sex hormone inducing the sex differentiation to male in decapod crustacean, although there is no report on the function of IAG peptide in vivo. In order to characterize P. virginalis IAG, we chemically synthesized it and evaluated its biological function in vivo. A and B chains were prepared by the ordinary solid-phase peptide synthesis, and three disulfide bonds were formed regioselectively by dimethyl sulfoxide oxidation, pyridylsulfenyl-directed thiolysis and iodine oxidation reactions. An IAG disulfide isomer was also prepared by the same manner. Circular dichroism spectral analysis revealed that the disulfide bond arrangement affected the peptide conformation, which was similar to the other insulin-family peptides analyzed so far. On the other hand, the glycan moiety attached at the B chain had no effect on the peptide secondary structure. Injection of the synthetic IAG and its disulfide isomer to female crayfish did not induce male characteristics on the external morphology, but both peptides suppressed the oocyte maturation in vivo. These results suggest that IAG has a pivotal role on the suppression of female secondary sex characteristics.

Genome and transcriptome assemblies of the kuruma shrimp, Marsupenaeus japonicus.

The kuruma shrimp Marsupenaeus japonicus (order Decapoda, family Penaeidae) is an economically important crustacean that occurs in shallow, warm seas across the Indo-Pacific. Here, using a combination of Illumina and Oxford Nanopore Technologies platforms, we produced a draft genome assembly of M. japonicus (1.70 Gbp; 18,210 scaffolds; scaffold N50 = 234.9 kbp; 34.38% GC, 93.4% BUSCO completeness) and a complete mitochondrial genome sequence (15,969 bp). As with other penaeid shrimp genomes, the M. japonicus genome is extremely rich in simple repeats, which occupies 27.4% of the assembly. A total of 26,381 protein-coding gene models (94.7% BUSCO completeness) were predicted, of which 18,005 genes (68.2%) were assigned functional description by at least one method. We also produced an Illumina-based transcriptome shotgun assembly (40,991 entries; 93.0% BUSCO completeness) and a PacBio Iso-Seq transcriptome assembly (25,415 entries; 67.5% BUSCO completeness). We envision that the M. japonicus genome and transcriptome assemblies will serve as useful resources for the basic research, fisheries management, and breeding programs of M. japonicus.

Sex Determination and Differentiation in Decapod and Cladoceran Crustaceans: An Overview of Endocrine Regulation.

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.

Methyl farnesoate regulatory mechanisms underlying photoperiod-dependent sex determination in the freshwater crustacean Daphnia magna.

Freshwater zooplankton Daphnia magna has been widely used in ecotoxicology studies. During the last 20 years, it has been demonstrated that the topical application of juvenile hormone (JH) or JH analogs to mother daphnids induce male offspring production. Based on this finding, an in vivo screening validation method for chemicals with JH agonistic effect has developed. Although this screening system successfully identified a number of JH-like chemicals, molecular mechanisms underlying the male sex-determining process remain largely unknown. To address this issue, we established a reliable male- or female-producing system using Daphnia pulex WTN6 strain by changing the rearing photoperiod. Taking advantage of this rearing system, we successfully found several factors involving male sex determination such as ionotropic glutamate receptors, protein kinase C and pantothenate. Here, we used two D. magna strains that can also control the production of female or male offspring by photoperiod differences as model species for ecotoxicology studies. We demonstrated that either treatment of antagonist of ionotropic glutamate receptors or inhibitor of protein kinase C strongly suppressed male offspring production even under male-producing conditions. Moreover, we revealed that male sex-determining processes are likely diverged between D. magna and D. pulex based on the current experiment. This study provides a fine experimental method for in vivo screening not only JH agonists but also JH antagonists. Moreover, using daphnids with photoperiod-dependent sex determination manner will hugely contribute to understanding the mode-of-action of JH in daphnids.

Impacts of Methyl Farnesoate and 20-Hydroxyecdysone on Larval Mortality and Metamorphosis in the Kuruma Prawn Marsupenaeus japonicus.

Physiological functions of juvenile hormone (JH) and molting hormone have been demonstrated in insects. JH, molting hormone and their mimics (insect growth regulators, IGRs) show endocrine-disrupting effects not only on target pest insects but also on other arthropod species such as crustaceans. However, little is known about the endocrine-disrupting effects of IGRs on benthic crustaceans. In this study, laboratory experiments were conducted to investigate effects of representative innate JH in crustaceans (methyl farnesoate, MF) and molting hormone (20-hydroxyecdysone, 20E, active form of ecdysteroid) on larval stages of the kuruma prawn Marsupenaeus japonicus, which is a decapod crustacean living in warm seawater. Larval development of kuruma prawn progresses in the order of nauplius, zoea, mysis, and then post-larvae with molting and metamorphosis, but it is unknown whether both MF and 20E have crucial roles in metamorphosis and molting of this species. Treatments of either MF or 20E on shrimp larvae were attempted at each developmental stage and those effects were validated. In terms of EC50 values between mortality and metamorphosis, there were apparent differences in the transition from nauplius to zoea (MF: 7.67 and 0.12 µM; 20E: 3.84 and 0.06 µM in survival and metamorphic rates, respectively). In contrast, EC50 values in MF and 20E treatments showed high consistency in the transitions between zoea to mysis (EC50 values for survival; MF: 1.25 and 20E: 0.22 µM), and mysis to post-larvae (EC50 values for survival; MF: 0.65 and 20E: 0.46 µM). These data suggest that nauplius has strong resistance against exposure to MF and 20E. Moreover, both chemicals induced high mortality triggered by the disruption of molting associated with metamorphosis. To our knowledge, this is the first experimental evidence that investigates in vivo physiological functions of MF and 20E in the larval stages of kuruma prawn, shedding light on not only ecotoxicological impacts of IGRs released into nature, but also endocrine mechanisms underlying larval development with metamorphosis in benthic decapod crustaceans.

Chemical synthesis of the crustacean insulin-like peptide with four disulfide bonds.

Among the insulin-family peptides, two additional cysteine residues other than six conserved cysteines are sometimes found in invertebrate insulin-like peptides (ILPs), although the synthetic method for such four disulfide ILPs has not yet been well established. In this study, we synthesized a crustacean insulin-like androgenic gland factor with four disulfides by the regioselective disulfide bond formation reactions using four orthogonal Cys-protecting groups. Its disulfide isomer could be also synthesized by the same method, indicating that the synthetic strategy developed in this study might be useful for the synthesis of other four disulfide ILPs.

Characterization of distinct ovarian isoform of crustacean female sex hormone in the kuruma prawn Marsupenaeus japonicus.

The eyestalk hormone, crustacean female sex hormone (CFSH), regulates the development of female secondary sexual characteristics in the blue crab Callinectes sapidus. After its discovery, several CFSH gene orthologs have been identified in some species of the suborder Pleocyemata as well. Similarly, in species of another suborder (Dendrobranchiata), an ortholog (Maj-CFSH) has been characterized as an eyestalk factor expressed in both females and males of the kuruma prawn, Marsupenaeus japonicus. In this study, another novel CFSH isoform was identified in the same species using cDNA cloning, expression analysis, and recombinant protein production. The isoform has "CFSH-family" structural characteristics but is dominantly expressed in the ovary, and was therefore designated as Maj-CFSH-ov. Its mRNA and protein levels in vitellogenic ovaries are higher than those in non-vitellogenic ovaries. In the vitellogenic ovary, both mRNA and protein expression of Maj-CFSH-ov are localized to oogonia and previtellogenic oocytes that occupy a small portion of vitellogenic ovaries, but not to the major developing oocytes. A vitellogenesis-inhibiting peptide of M. japonicus (Pej-SGP-I) reduced the expression of vitellogenin in incubated ovarian fragments, but not that of Maj-CFSH-ov. These results indicate that M. japonicus possesses two CFSH isoforms that are derived from distinct tissues, the central X-organ/sinus gland complex and peripheral ovaries. The expression profile of Maj-CFSH-ov suggests its involvement in some reproductive process other than vitellogenesis.

Direct evidence for the function of crustacean insulin-like androgenic gland factor (IAG): total chemical synthesis of IAG.

Insulin-like androgenic gland factor (IAG) is presumed to be a sex differentiation factor so-called androgenic gland hormone (AGH) in decapod crustacean, although the function of IAG peptide has not yet been reported. In this study, we synthesized IAG from the prawn, Marsupenaeus japonicus, and its function was assessed by an in vitro bioassay. As a result, IAG with the insulin-type disulfide bond arrangement showed biological activity, whereas its disulfide isomer did not. These results strongly suggest that the native IAG peptide has an insulin-type disulfide, and it is the decapod AGH.

Identification and characterisation of a calcium carbonate-binding protein, blue mussel shell protein (BMSP), from the nacreous layer.

The nacreous layer of molluscan shells consists of a highly organised, layered structure comprising calcium carbonate aragonite crystals, each surrounded by an organic matrix. In the Japanese pearl oyster Pinctada fucata, the Pif protein from the nacreous layer functions in aragonite binding, and plays a key role in nacre formation. Here, we investigated whether the blue mussel Mytilus galloprovincialis also has a protein with similar functions in the nacreous layer. By using a calcium carbonate-binding assay, we identified the novel protein blue mussel shell protein (BMSP) 100 that can bind calcium carbonate crystals of both aragonite and calcite. When the entire sequence of a cDNA encoding BMSP 100 was determined, it was found that BMSP is a preproprotein consisting of a signal peptide and two proteins, BMSP 120 and BMSP 100. BMSP 120 contains four von Willebrand factor A (VWA) domains and one chitin-binding domain, thus suggesting that it has a role in maintaining structure within the matrix. Immunohistochemical analysis revealed that BMSP 100 is present throughout the nacreous layer with dense localisation in the myostracum. Posttranslational modification analysis indicated that BMSP 100 is phosphorylated and glycosylated. These results suggest that there is a common molecular mechanism between P. fucata and M. galloprovincialis that underlies the nacreous layer formation.

Correct disulfide pairing is required for the biological activity of crustacean androgenic gland hormone (AGH): synthetic studies of AGH.

Androgenic gland hormone (AGH) of the woodlouse, Armadillidium vulgare, is a heterodimeric glycopeptide. In this study, we synthesized AGH with a homogeneous N-linked glycan using the expressed protein ligation method. Unexpectedly, disulfide bridge arrangement of a semisynthetic peptide differed from that of a recombinant peptide prepared in a baculovirus expression system, and the semisynthetic peptide showed no biological activity in vivo. To confirm that the loss of biological activity resulted from disulfide bond isomerization, AGH with a GlcNAc moiety was chemically synthesized by the selective disulfide formation. This synthetic AGH showed biological activity in vivo. These results indicate that the native conformation of AGH is not the most thermodynamically stable form, and correct disulfide linkages are important for conferring AGH activity.

A convenient method for preparation of biologically active recombinant CHH of the kuruma prawn, Marsupenaeus japonicus, using the bacterial expression system.

Crustacean hyperglycemic hormone (CHH) not only plays an important role in the modulation of hemolymph glucose level but also functions in other biological events including molting, reproduction and stress response. Of the six CHHs characterized in Marsupenaeus japonicus, an expression system for recombinant Pej-SGP-VII (rPej-SGP-VII-amide) has not yet been established. Here, we established a procedure using a Nus-tag for solubilization, thereby soluble and biologically active rPej-SGP-VII-amide could successfully be obtained by a simpler procedure than previous ones used for producing other recombinant Pej-SGPs (Pej-SGP-I, III and IV). It was found that rPej-SGP-VII-amide thus obtained had the correct arrangement of intramolecular disulfide bonds and helix-rich secondary structure. The established expression system for rPej-SGP-VII-amide may be applicable for the preparation of other recombinant CHHs.

High-resolution computed tomographic findings of small peripherally located squamous cell carcinoma.

With the spread of high-resolution computed tomography (HRCT) screening for lung cancer, we are increasingly faced with the need to determine whether certain small lesions are benign or malignant. The features of small adenocarcinomas have been clarified but not those of squamous cell carcinoma. The objective of our study was therefore to clarify the HRCT findings of peripherally located squamous cell carcinomas less than 2 cm in maximum dimension. Subjects consisted of 27 consecutive pathologically proven cases of peripherally located squamous cell carcinoma that were less than 2 cm in maximum dimension. HRCT findings of all 27 cases were analyzed retrospectively and independently by three radiologists who were unaware of the pathological diagnosis, and decisions were reached by consensus with special attention to 10 review points. Internal characteristic features included calcification, cavity formation, and air bronchogram. Tumor margin features included spiculation, notching, irregularity, and ground-glass opacity. Surrounding structural features consisted of pleural indentation, pulmonary emphysema, and satellite lesions. The presence of irregularity (70.4%), surrounding pulmonary emphysema (70.4%), and pleural indentation (51.9%) was observed frequently. No mass was accompanied by calcification. HRCT images of peripherally located squamous cell carcinoma suggested that the demonstration of irregularity, surrounding pulmonary emphysema, pleural indentation, and absence of calcification may contribute to the accurate CT diagnosis of small peripheral squamous cell carcinoma.

Characterization and expression of the putative ovarian lipoprotein receptor in the Kuruma prawn, Marsupenaeus japonicus.

Ovarian low density lipoproteins (LDL) such as vitellogenin (Vg) are the precursors of the major yolk protein vitellin, and constitute the major source of nutrients serving the developing embryo. The objective of this study was to gain a better understanding of crustacean egg development by focusing on the process of Vg internalization by its receptor (ovarian LDLR). First, an ovarian LDLR cDNA sequence in Marsupenaeus japonicus was determined. Ovarian LDLR mRNA expression was then examined, and was seen to be specific to the ovary, exhibiting highest levels during the previtellogenic stage. This pattern of ovarian LDLR expression is thought to signify preparation for yolk protein incorporation into the oocyte. Using immunoblotting techniques, an ovarian LDLR band was detected whose size was similar to that estimated from the deduced amino acid sequence. The ovarian LDLR protein was expressed only at the onset of vitellogenesis, and histological studies supported these observations. This is the first occasion that the ovarian LDLR and its expression dynamics during vitellogenesis have been fully characterized in a crustacean.