Vargula Hilgendorfii Research Articles

Intraspecific nucleotide polymorphisms in seven complete sequences of mitochondrial DNA of the luminous ostracod, Vargula hilgendorfii (Crustacea, Ostracoda)

We demonstrated intraspecific nucleotide polymorphisms of mitochondrial DNA (mtDNA) of the luminescent marine ostracod, Vargula hilgendorfii, from seven different locations around Japan. The intraspecific identity of the complete mtDNA within each of these populations was 92.5%–99.7%, showing that nucleotide polymorphisms existed in their mtDNA. The characteristics of codon usage and amino acid composition were not uniform, despite being from the same species. However, these variations may not be random and may follow certain rules. The identities of some specific sites in these enzymes in mitochondria were less 80%, suggesting the possibility of adaptation of enzymatic function depending on the biogeographical regions. Our findings of a higher nucleotide substitution rate support monitoring mtDNA to track comparatively recent evolutionary events.

Laboratory culture of the California Sea Firefly Vargula tsujii (Ostracoda: Cypridinidae): Developing a model system for the evolution of marine bioluminescence

Bioluminescence, or the production of light by living organisms via chemical reaction, is widespread across Metazoa. Laboratory culture of bioluminescent organisms from diverse taxonomic groups is important for determining the biosynthetic pathways of bioluminescent substrates, which may lead to new tools for biotechnology and biomedicine. Some bioluminescent groups may be cultured, including some cnidarians, ctenophores, and brittle stars, but those use luminescent substrates (luciferins) obtained from their diets, and therefore are not informative for determination of the biosynthetic pathways of the luciferins. Other groups, including terrestrial fireflies, do synthesize their own luciferin, but culturing them is difficult and the biosynthetic pathway for firefly luciferin remains unclear. An additional independent origin of endogenous bioluminescence is found within ostracods from the family Cypridinidae, which use their luminescence for defense and, in Caribbean species, for courtship displays. Here, we report the first complete life cycle of a luminous ostracod (Vargula tsujii Kornicker & Baker, 1977, the California Sea Firefly) in the laboratory. We also describe the late-stage embryogenesis of Vargula tsujii and discuss the size classes of instar development. We find embryogenesis in V. tsujii ranges from 25–38 days, and this species appears to have five instar stages, consistent with ontogeny in other cypridinid lineages. We estimate a complete life cycle at 3–4 months. We also present the first complete mitochondrial genome for Vargula tsujii. Bringing a luminous ostracod into laboratory culture sets the stage for many potential avenues of study, including learning the biosynthetic pathway of cypridinid luciferin and genomic manipulation of an autogenic bioluminescent system.

Mechanistic Insight into Cypridina Bioluminescence with a Combined Experimental and Theoretical Chemiluminescent Approach.

The bioluminescent reaction of the "sea firefly" Cypridina hilgendorfii is a prototypical system for marine bioluminescence, as its substrate possesses an imidazopyrazinone core that is a common link among organisms of eight phyla. The elucidation of the mechanism behind Cypridina bioluminescence is essential for future applications in bioimaging, biomedicine, and bioanalysis. In this study we have investigated the key step of chemiexcitation with a combined experimental and theoretical approach. The obtained results indicate that neutral dioxetanone is responsible for efficient chemiexcitation, as the thermolysis of this species gives access to a long region of the potential energy surface (PES), where the ground and excited singlet states are degenerated. Contrary to expected, neither chemically induced electron-exchange luminescence (CIEEL) nor charge transfer-initiated luminescence (CTIL) can be used to explain imidazopyrazinone-based bioluminescence, as there is no clear relationship between electron (ET)/charge (CT) transfer (occurring between the electron-rich moiety and dioxetanone) and chemiexcitation. Attractive electrostatic interactions between the CO2 and oxyluciferin moieties allow neutral dioxetanone to spend time in the PES region of degeneracy, while repulsive interactions for anionic dioxetanone lead to a quicker CO2 detachment.

Mechanistic insight into marine bioluminescence: photochemistry of the chemiexcited Cypridina (sea firefly) lumophore.

Cypridina hilgendorfii (sea firefly) is a bioluminescent crustacean whose bioluminescence (BL) reaction is archetypal for a number of marine organisms, notably other bioluminescent crustaceans and coelenterates. Unraveling the mechanism of its BL is paramount for future applications of its strongly emissive lumophore. Cypridina produces light in a three-step reaction: First, the cypridinid luciferin is activated by an enzyme to produce a peroxide intermediate, cypridinid dioxetanone (CDO), which then decomposes to generate excited oxyluciferin (OxyCLnH*). Finally, OxyCLnH* deexcites to its ground state along with emission of bright blue light. Unfortunately, the detailed mechanism of the critical step, the thermolysis of CDO, remains unknown, and it is unclear whether the light emitter is generated from a neutral form (CDOH) or anionic form (CDO(-)) of the CDO precursor. In this work, we investigated the key step in the process by modeling the thermal decompositions of both CDOH and CDO(-). The calculated results indicate that the decomposition of CDO(-) occurs via the gradually reversible charge transfer (CT)-initiated luminescence (GRCTIL) mechanism, whereas CDOH decomposes through an entropic trapping mechanism without an obvious CT process. The thermolysis of CDO(-) is sensitive to solvent effects and is energetically favorable in polar environments compared with the thermolysis of CDOH. The thermolysis of CDO(-) produces the excited oxyluciferin anion (OxyCLn(-)*), which combines with a proton from the environment to form OxyCLnH*, the actual light emitter for the natural system.

Mortality of juvenile Japanese flounder Paralichthys olivaceus caused by scavenging amphipods and ostracods in trap experiments

Trap experiments were conducted using wild and artificially reared juvenile Japanese flounder in various conditions as baits in a flounder nursery habitat of Mano Bay. All of the live wild flounder placed in the traps with sand survived in good health. However, most of the live wild flounder in the traps without sand and live-reared flounder in the traps with and without sand, as well as injured and dead-reared flounder, were consumed and stripped to skeletons overnight. A large number of the lysianassoid Orchomene sp. and Orchomene naikaiensis, and the cypridinid Vargula hilgendorfii, which are typical scavengers, accumulated in the traps with consumed flounder; however, few amphipods and ostracods occurred in traps without consumed flounder. Thus, once flounder are detected and reached by a swarm of these crustaceans, flounder may be preyed on, irrespective of the condition of the fish. This finding is supported by aquarium observations that amphipods and ostracods were able to kill and consume live flounder. Burial in sand will potentially prevent the crustaceans from detecting wild flounder by interfering with the odor from the fish. The results of our study raise the possibility that scavenging crustaceans are able to predate hatchery-reared flounder released in the sea.

Evolution of the complement system in protostomes revealed by de novo transcriptome analysis of six species of Arthropoda

To elucidate the evolutionary history of the complement system in Arthropoda, de novo transcriptome analysis was performed with six species among the Chelicerata, Myriapoda, and Crustacea, and complement genes were identified based on their characteristic domain structures. Complement C3 and factor B (FB) were identified from a sea spider, a jumping spider, and a centipede, but not from a sea firefly or two millipede species. No additional complement components identifiable by their characteristic domain structures were found from any of these six species. These results together with genome sequence information for several species of the Hexapoda suggest that the common ancestor of the Arthropoda possessed a simple complement system comprising C3 and FB, and thus resembled the alternative pathway of the mammalian complement system. It was lost at least twice independently during the evolution of Arthropoda in the millipede lineage and in the common ancestor of Crustacea and Hexapoda.

ChemInform Abstract: Identification of 3‐Enol Sulfate of Cypridina Luciferin, Cypridina Luciferyl Sulfate, in the Sea‐Firefly Cypridina (Vargula) hilgendorfii.

Abstract Cypridina luciferin from the luminous ostracod Cypridina (Vargula) hilgendorfii has an imidazopyrazinone core structure (3,7-dihydroimidazopyrazin-3-one), which is identical to that of coelenterazine. Cypridina luciferyl sulfate (3-enol sulfate of Cypridina luciferin) was isolated for the first time and the chemical structure was identified by LC/ESI–TOF–MS analysis. Furthermore, Cypridina luciferyl sulfate was chemically synthesized, and its absorption and MS/MS spectra were in agreement with that of Cypridina luciferyl sulfate isolated. Using the crude extracts of Cypridina specimens, Cypridina luciferyl sulfate could be converted to Cypridina luciferin in the presence of adenosine 3′,5′-diphosphate (PAP), and Cypridina luciferin was converted to Cypridina luciferyl sulfate in the presence of 3′-phosphoadenosine 5′-phosphosulfate (PAPS). These results suggested that a sulfotransferase catalyzes the reversible sulfation of Cypridina luciferin in Cypridina hilgendorfii. In aqueous solution, Cypridina luciferyl sulfate was more stable than Cypridina luciferin and might be a storage form of Cypridina luciferin.

Identification of 3-enol sulfate of Cypridina luciferin, Cypridina luciferyl sulfate, in the sea-firefly Cypridina (Vargula) hilgendorfii

Cypridina luciferin from the luminous ostracod Cypridina (Vargula) hilgendorfii has an imidazopyrazinone core structure (3,7-dihydroimidazopyrazin-3-one), which is identical to that of coelenterazine. Cypridina luciferyl sulfate (3-enol sulfate of Cypridina luciferin) was isolated for the first time and the chemical structure was identified by LC/ESI–TOF–MS analysis. Furthermore, Cypridina luciferyl sulfate was chemically synthesized, and its absorption and MS/MS spectra were in agreement with that of Cypridina luciferyl sulfate isolated. Using the crude extracts of Cypridina specimens, Cypridina luciferyl sulfate could be converted to Cypridina luciferin in the presence of adenosine 3′,5′-diphosphate (PAP), and Cypridina luciferin was converted to Cypridina luciferyl sulfate in the presence of 3′-phosphoadenosine 5′-phosphosulfate (PAPS). These results suggested that a sulfotransferase catalyzes the reversible sulfation of Cypridina luciferin in Cypridina hilgendorfii. In aqueous solution, Cypridina luciferyl sulfate was more stable than Cypridina luciferin and might be a storage form of Cypridina luciferin.

Triple Bioluminescence Imaging for In Vivo Monitoring of Cellular Processes

Bioluminescence imaging (BLI) has shown to be crucial for monitoring in vivo biological processes. So far, only dual bioluminescence imaging using firefly (Fluc) and Renilla or Gaussia (Gluc) luciferase has been achieved due to the lack of availability of other efficiently expressed luciferases using different substrates. Here, we characterized a codon-optimized luciferase from Vargula hilgendorfii (Vluc) as a reporter for mammalian gene expression. We showed that Vluc can be multiplexed with Gluc and Fluc for sequential imaging of three distinct cellular phenomena in the same biological system using vargulin, coelenterazine, and D-luciferin substrates, respectively. We applied this triple imaging system to monitor the effect of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) delivered using an adeno-associated viral vector (AAV) on brain tumors in mice. Vluc imaging showed efficient sTRAIL gene delivery to the brain, while Fluc imaging revealed a robust antiglioma therapy. Further, nuclear factor-κB (NF-κB) activation in response to sTRAIL binding to glioma cells death receptors was monitored by Gluc imaging. This work is the first demonstration of trimodal in vivo bioluminescence imaging and will have a broad applicability in many different fields including immunology, oncology, virology, and neuroscience.

大阪湾東岸域におけるウミホタルの生息に適した底層環境と影響する周辺特性

大阪湾東岸域におけるウミホタルの生息に適した底層環境と影響する周辺特性

Based on a review of the data, use of the term ‘cypridinid’ solves the Cypridina/Vargula dilemma for naming the constituents of the luminescent system of ostracods in the family Cypridinidae

AbstractThis issue's featured article; Based on a review of the data, use of the term ‘cypridinid’ solves the Cypridina/Vargula dilemma for naming the constituents of the luminescent system of ostracods in the family Cypridinidae is authored by Jim Morin of Cornell University.The cover graphic indicates the evolutionary relationships among the luminescent and non‐luminescent species within the well‐known cypridinid ostracods including the Japanese species Vargula hilgendorfii, formerly known as Cypridina hilgendorfii, from which the components of this luminescent system were identified, including the cypridinid luciferin shown in the upper left. Because all luminescent members of the clade use the same basic components, to avoid confusion, it is most parsimonious to use the term ‘cypridinid’ rather than generic designations. Based on the phylogeny it can be inferred that luminescence first evolved as a predator deterrent and secondarily, but only in the Caribbean, as courtship signals. Only about half the species in the family are luminescent. Of the luminescent species, all probably use their light to deter predators while about half (the Caribbean species) also use their light in sexual reproduction. The family includes three recently described new luminescent genera and at least four undescribed luminescent genera (and probably several more). Several species (A‐F) are shown on the right including: A: Kornickeria n. sp.; B. H‐Group (new undescribed genus) n. sp.; C. Photeros morini; D. ‘Vargula’ hilgendorfii (note: this species belongs in a new undescribed genus separate from both Vargula and Cypridina); E. Cypridina dentata and F. Skogsbergia lerneri (a non‐luminescent species). See cited references 11–13 and references therein for details.

Based on a review of the data, use of the term ‘cypridinid’ solves theCypridina/Varguladilemma for naming the constituents of the luminescent system of ostracods in the family Cypridinidae

The luciferin of the ostracod Vargula hilgendorfii (formerly Cypridina hilgendorfii) is often termed Cypridina luciferin in the scientific literature, but, to avoid ambiguity and based on a review of the literature, is best referred to by the more inclusive term cypridinid luciferin. This situation exemplifies how new knowledge in systematics and the resulting nomenclatural changes can result in evolutionary insights. Similar solutions can probably be applied to other taxonomic dilemmas.

Embryonic development clarifies polyphyly in ostracod crustaceans

AbstractThe present study describes the embryonic developmental process of the bioluminescent ostracod crustacean Vargula hilgendorfii. Optical microscopy, scanning electron microscopy, DAPI staining and video recording were used for observations. This study is the first detailed report of the embryonic development of a myodocopid ostracod. Contrary to previous studies, cleavage occurred in the yolk sphere and no evident cleavage furrow was found. No nauplius stage was found, and five pairs of appendages developed simultaneously. A bivalved carapace developed from two independent buds of the carapace valves. The buds of the left and right valves are enlarged, and become combined. The combined ‘one‐piece’ carapace was divided by the formation of a hinge, and the usual bivalved carapace was formed. On the 16th day, embryos hatched as juveniles with six pairs of appendages, a pair of immature appendages, a pair of compound eyes, a median eye and a bivalved carapace. An important suggestion for the classification of Ostracoda is derived from the observed development of appendages and carapace, because the subclass Ostracoda is defined mainly by the similarities of appendages and the bivalved carapace. The present observations clearly show that the developmental process of Myodocopa differs from that of Podocopa, and supports polyphyly of the Ostracoda.

サイズヒストグラムによるウミホタル個体群動態の解析

The life history of the myodocopid ostracod Vargula hilgendorfii was investigated by size-frequency distribution analysis in Akita Port, Japan, from April to December, 2003. Vargula hilgendorfii individuals were collected using baited traps, and the carapace length was measured by computer analysis of a digital photograph of each individual taken under a stereomicroscope. The population was divided into seven groups based on carapace length. These groups corresponded to the five juvenile stages, the adult male stage, and the adult female stage. Each peak in the number of V. hilgendorfii individuals is related to the growth of the population. Our field data clearly suggest that the Akita Port population of V. hilgendorfii produces two generations per year. Adults that survived the winter became sexually active in May and released free-living juveniles in July. After reproducting, they died without having entered a second reproductive cycle. The first-generation juveniles reached the adult stage after five molts and released second-generation juveniles in September. By winter, these second-generation juveniles reached maturity without releasing any new juveniles; they become sexually active in the following spring.

Generation and distribution of reactive oxygen species in the skin of hairless mice under UVA: studies on in vivo chemiluminescent detection and tape stripping methods

Although the formation of reactive oxygen species (ROS) in the skin induced by the ultraviolet (UV) light has been shown to lead to many cutaneous disorders, skin cancer and photoageing, the mechanism and distribution of ROS generation has not yet been definitively determined. In the present study, we examined the distribution of UVA-induced ROS in the skin of live hairless mice, using our proposed in vivo imaging chemiluminescent (CL) method to detect ROS combined with a CL probe (cypridina hilgendorfii luciferin analogue; CLA) and tape stripping (TS) technique. The CL intensities in the skin of live hairless mice were confirmed to significantly increase by UVA exposure. When TS was conducted five times in a maximum level after CL measurement following UVA exposure and subsequent CLA application, CL intensities due to UVA-induced ROS generation in the residual skin decreased to 10% of the original levels; and those in the stripped skin on each tape decreased in the stripped order such as 52%, 16%, 11%, 6% and 5%. Next, CLA was applied and then CL intensities were measured in the residual skin after advance 1, 3 and 5 tape strippings, and CL intensities due to ROS were detected primarily in the outer layer of the skin. On the basis of these results, we concluded that ROS induced by UVA exposure occurs and distributes in the outermost layer of the stratum corneum.

Concerted Evolution of Duplicated Control Regions within an Ostracod Mitochondrial Genome

The luminescent marine ostracod Vargula hilgendorfii comprises distinct populations around the Japanese islands. Its mitochondrial DNA is unusual, with duplicated control regions (CRs; CR#1 and CR#2). We determined the sequences of ostracod CRs in 7 different populations. The sequences of CR#1 and CR#2 within any population were extremely similar, above 99.7%; moreover, their derived evolutionary tree indicates that the pairs of CRs have evolved in concert within each mitochondrial genome. These results suggest that an exact replication mechanism controls the concerted evolution of CRs.

New reporter system for Per1 and Bmal1 expressions revealed self‐sustained circadian rhythms in peripheral tissues

A new reporter system for monitoring expressions of two clock genes, Per1 and Bmal1, from a single tissue in culture was developed in mice. Reporters are Vargula hilgendorfii luciferase (VL) and firefly luciferase (FL), whose activities are increased in parallel with Per1 and Bmal1 expressions, respectively. Formal properties of the circadian system in transgenic mice are indistinguishable from those in wild-type animals. Circadian rhythms in Per1-VL and Bmal1-FL in the suprachiasmatic nucleus (SCN) were robust and anti-phasic, although they were phase delayed by 4-8 h as compared with circadian rhythms in respective transcript levels in vivo. In peripheral tissues such as liver, circadian rhythms in Bmal1-FL persisted for more than 3 weeks. In the course of prolonged culture, circadian rhythms apparently damped out, but were restored immediately by refreshment of the culture medium. Restoration of the circadian rhythm is unlikely to be due to resetting of desynchronized population oscillation, because peripheral circadian rhythms did not show a type 0 phase response curve (PRC) for medium refreshment, a requirement for instantaneous resetting of circadian oscillation. Long-term persistence of circadian oscillation in spite of external perturbations supports an idea that circadian oscillations in peripheral tissues are self-sustained.

Stereoselective Incorporation of Isoleucine into Cypridina Luciferin inCypridina hilgendorfii(Vargula hilgendorfii)

The emission of light in the marine ostracod Cypridina hilgendorfii (presently Vargula hilgendorfii) is produced by the Cypridina luciferin-luciferase reaction in the presence of molecular oxygen. Cypridina luciferin has an asymmetric carbon derived from isoleucine, and the absolute configuration is identical to the C-3 position in L-isoleucine or D-alloisoleucine. To determine the stereoselective incorporation of the isoleucine isomers (L-isoleucine, D-isoleucine, L-alloisoleucine, and D-alloisoleucine), we synthesized four (2)H-labeled isoleucine isomers and examined their incorporation into Cypridina luciferin by feeding experiments. Judging by these results, L-isoleucine is predominantly incorporated into Cypridina luciferin. This suggests that the isoleucine unit of Cypridina luciferin is derived from L-isoleucine, but not from D-alloisoleucine.

Biogeography of Luminous Marine Ostracod Driven Irreversibly by the Japan Current

The biogeography of the luminous marine ostracod Vargula hilgendorfii, also called "Umihotaru," shows that this organism may have arrived relatively recently on the Japanese islands during the final glacier period approximately 10,000 years ago. Phylogenetic relationships also strongly indicate that the Japan Current drove the Umihotaru ostracod northward. It is evident that the Umihotaru ostracod spread rapidly to the major Japanese islands 3,000 km north, whereas its spread was slow in the southwest of the Japanese islands, covering a distance of 400 km. The meandering of the Japan Current, where it passes by the Tokara Gap at 28 degrees N latitude, may be a barrier to Umihotaru ostracod extension.

Differential Expression of Duplicated Opsin Genes in Two EyeTypes of Ostracod Crustaceans

In the first molecular study of ostracod (Crustacea) vision, we present partial cDNA sequences of ostracod visual pigment genes (opsins). We found strong support for differential expression of opsins in ostracod median and compound eyes and suggest that photoreceptor specific expression may be a general phenomenon in organisms with multiple receptors. We infer that eye-specific expression predates the divergence of the two species examined, Skogsbergia lerneri and Vargula hilgendorfii, because eye-specific opsin orthologs are present in both species. We found multiple opsin loci in ostracods, estimating that at least eight are present in Skogsbergia lerneri. All opsins from both ostracod species examined are more closely related to each other than to any other known opsin sequences. Because we find no evidence for gene conversion or alternative splicing, we suggest the occurrence of many recent gene duplications. Why ostracods may have retained multiple recent opsin gene duplicates is unknown, but we discuss several possible hypotheses.