Chamberlinius Hualienensis Research Articles

Morphological and molecular characterization of Travassosinema viatorum n. sp. (Nematoda: Oxyuridomorpha: Travassosinematidae) from the invasive millipede Chamberlinius hualienensis Wang, 1956 (Diplopoda: Polydesmida: Paradoxosomatidae) in Okinawa, Japan.

Travassosinema viatorum n. sp. (Nematoda: Oxyuridomorpha: Travassosinematidae) is described from the invasive millipede Chamberlinius hualienensis Wang, 1956 (Diplopoda: Polydesmida: Paradoxosomatidae) in Okinawa, Japan. Females of T. viatorum n. sp. resemble T. claudiae Morffe & Hasegawa, 2017; T. thyropygi Hunt, 1996 and T. travassosi Rao, 1958 by lacking both lateral alae and a contraction posterior to the vulva. The body length of T. viatorum n. sp. is shorter than the aforementioned species. It differs from T. claudiae and T. thyropygi by its comparatively longer oesophagus, more posterior vulva, lack of cuticular ribs in the alae of the umbraculum and in the position of the nerve ring and excretory pore. Travassosinema viatorum n. sp. can be differentiated from T. travassosi by the extension of the cephalic umbraculum and the smaller eggs. The phylogeny of T. viatorum n. sp. is inferred by the D2-D3 domains of the 28S rDNA. The new species is located in a monophyletic clade with other sequences of Travassosinema from millipedes. The comparison of the phylogenies of Travassosinema and their hosts points to host switching rather than coevolution as a mechanism to explain the host-nematode relationships. This constitutes the second species of the genus Travassosinema described from the Japanese archipelago and the first oxyuridomorph nematodes found in C. hualienensis.

Initial defensive secretory compounds emitted from the live millipede and the induction of apoptotic cell death

The initial defensive secretory compounds emitted from a live millipede have not yet been clarified. This study focused on elucidating the initial secretory compounds emitted from a live millipede. Pre-concentration of the defensive secretory volatile organic compounds (VOC) from the live Polidesmida millipedes, Chamberlinius hualienensis and Oxidus gracilis, was performed using a three-stage VOC concentration technique by an on-line GC/MS system. As a result, the monoterpenes derived from the plant metabolite; i.e., α-pinene, α-thujene, β-pinene, 3-carene, β-myrcene, β-phellandrene, γ-terpinene, o,m,p-cymenes, limonene and camphene were first detected as the initial secretory substances. It was elucidated that some plant monoterpenes have a repellent effect and antifungal and antibacterial actions which are used as defensive substances. In addition, this study also confirmed that these monoterpenes induced apoptotic cell death involved in the induction of the caspase 3/7 activity. The millipede feeds on fallen or withered leaves containing the monoterpenes. Thus, the millipede accumulates the plant defensive secretions in the exocrine defense glands of the body somites, which would be used as against predators.

Discovery and Structural Analysis to Improve the Enantioselectivity of Hydroxynitrile Lyase from Parafontaria laminata Millipedes for (R)-2-Chloromandelonitrile Synthesis.

Hydroxynitrile lyase (HNL) catalyzes the reversible synthesis and degradation of cyanohydrins, which are important synthetic intermediates for fine chemical and pharmaceutical industries. Here, we report the discovery of HNL from Parafontaria laminata (PlamHNL) millipedes, purification of the HNL to homogeneity, expression of the gene for the enzyme in heterologous expression hosts, and increase in the reaction rate and enantioselectivity in the synthesis of 2-chloromandelonitrile by protein engineering. The recombinant PlamHNL expressed in Pichia pastoris is glycosylated and has a higher thermostability and pH stability than the nonglycosylated HNL expressed in Escherichia coli. PlamHNL showed a unique wide substrate specificity among other millipede HNLs acting on various cyanohydrins, including 2-chloromandelonitrile, a key intermediate for the antithrombotic agent clopidogrel. We solved the X-ray crystal structure of the PlamHNL and found that the catalytic residues were almost identical to those of HNL from Chamberlinius hualienensis, although the forming binding cavity was different. In order to improve the catalytic activity and stereoselectivity, a computational structure-guided directed evolution approach was performed by an enzyme–substrate docking simulation at all of the residues that were exposed on the surface of the active site. The PlamHNL-N85Y mutant showed higher conversion (91% conversion with 98.2% ee of the product) than the wild type (76% conversion with 90% ee of the product) at pH 3.5 and 25 °C for 30 min of incubation. This study shows the diversity of millipede HNLs and reveals the molecular basis for improvement of the activity and stereoselectivity of the wild-type HNL to increase the reaction rate and enantioselectivity in the synthesis of 2-chloromandelonitrile.

R-hydroxynitrile lyase from the cyanogenic millipede, Chamberlinius hualienensis-A new entry to the carrier protein family Lipocalines.

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrin into cyanide and the corresponding aldehyde or ketone. Moreover, they catalyze the synthesis of cyanohydrin in the reverse reaction, utilized in industry for preparation of enantiomeric pure pharmaceutical ingredients and fine chemicals. We discovered a new HNL from the cyanogenic millipede, Chamberlinius hualienensis. The enzyme displays several features including a new primary structure, high stability, and the highest specific activity in (R)-mandelonitrile ((R)-MAN) synthesis (7420 U·mg-1 ) among the reported HNLs. In this study, we elucidated the crystal structure and reaction mechanism of natural ChuaHNL in ligand-free form and its complexes with acetate, cyanide ion, and inhibitors (thiocyanate or iodoacetate) at 1.6, 1.5, 2.1, 1.55, and 1.55Å resolutions, respectively. The structure of ChuaHNL revealed that it belongs to the lipocalin superfamily, despite low amino acid sequence identity. The docking model of (R)-MAN with ChuaHNL suggested that the hydroxyl group forms hydrogen bonds with R38 and K117, and the nitrile group forms hydrogen bonds with R38 and Y103. The mutational analysis showed the importance of these residues in the enzymatic reaction. From these results, we propose that K117 acts as a base to abstract a proton from the hydroxyl group of cyanohydrins and R38 acts as an acid to donate a proton to the cyanide ion during the cleavage reaction of cyanohydrins. The reverse mechanism would occur during the cyanohydrin synthesis. (Photo: Dr. Yuko Ishida) DATABASES: Structural data are available in PDB database under the accession numbers 6JHC, 6KFA, 6KFB, 6KFC, and 6KFD.

Effects of codon optimization and glycosylation on the high-level production of hydroxynitrile lyase from Chamberlinius hualienensis in Pichia pastoris.

A hydroxynitrile lyase (HNL) from the millipede Chamberlinius hualienensis has high potential for industrial use in the synthesis of cyanohydrins. However, obtaining sufficient amounts of millipedes is difficult, and the production of the Chamberlinius hualienensis HNL (ChuaHNL) in E. coli has not been very successful. Therefore, we investigated the conditions required for high-yield heterologous production of this enzyme using Pichia pastoris. When we employed P. pastoris to express His-ChuaHNL, the yield was very low (22.6 ± 3.8U/L culture). Hence, we investigated the effects of ChuaHNL codon optimization and the co-production of two protein disulfide isomerases (PDIs) [from P. pastoris (PpPDI) and C. hualienensis (ChuaPDI1, ChuaPDI2)] on His-ChuaHNL production. The productivity of His-ChuaHNL was increased approximately 140 times per unit culture to 3170 ± 144.7U/L by the co-expression of codon-optimized ChuaHNL and PpPDI. Moreover, we revealed that the N-glycosylation on ChuaHNL had a large effect on the stability, enzyme secretion, and catalytic properties of ChuaHNL in P. pastoris. This study demonstrates an economical and efficient approach for the production of HNL, and the data show that glycosylation has a large effect on the enzyme properties and the P. pastoris expression system.

Hydroxynitrile lyases from cyanogenic millipedes: molecular cloning, heterologous expression, and whole-cell biocatalysis for the production of (R)-mandelonitrile

Hydroxynitrile lyases (HNLs), which are key enzymes in cyanogenesis, catalyze the cleavage of cyanohydrins into carbonyl compounds and hydrogen cyanide. Since HNLs also catalyze the reverse reaction, they are used industrially for the asymmetric synthesis of cyanohydrins, which are valuable building blocks of pharmaceuticals and fine chemicals. HNLs have been isolated from cyanogenic plants and bacteria. Recently, an HNL from the cyanogenic millipede Chamberlinius hualienensis was shown to have the highest specific activity for (R)-mandelonitrile synthesis, along with high stability and enantioselectivity. However, no HNLs have been isolated from other cyanogenic millipedes. We identified and characterized HNLs from 10 cyanogenic millipedes in the Paradoxosomatidae and Xystodesmidae. Sequence analyses showed that HNLs are conserved among cyanogenic millipedes and likely evolved from one ancestral gene. The HNL from Parafontaria tonominea was expressed in Escherichia coli SHuffle T7 and showed high specific activity for (R)-mandelonitrile synthesis and stability at a range of pHs and temperatures. The stability of millipede HNLs is likely due to disulfide bond(s). The E. coli cells expressing HNL produced (R)-mandelonitrile with 97.6% enantiomeric excess without organic solvents. These results demonstrate that cyanogenic millipedes are a valuable source of HNLs with high specific activity and stability.

Two nematodes (Nematoda: Diplogastridae, Rhabditidae) from the invasive millipede Chamberlinius hualienensis Wang, 1956 (Diplopoda, Paradoxosomatidae) on Hachijojima Island in Japan.

Abstract Millipedes may cause unexpected damage when they are introduced to new locations, becoming invaders that leave behind their old parasites and predators. Therefore, it was interesting to find numerous rhabditid nematodes within the gut of the invasive phytophagous millipede Chamberlinius hualienensis Wang, 1956 (Diplopoda, Paradoxosomatidae) from Hachijojima (Japan) in November, 2014. This millipede originated in Taiwan but was discovered in Japan in 1986. The nematodes were identified as juvenile Oscheius rugaoensis (Zhang et al., 2012) Darsouei et al., 2014 (Rhabditidae), and juvenile and adult Mononchoides sp. (Diplogastridae) based on images, morphometrics, and sequences of 18S and 28S rDNA. A novel short 28S sequence of a separate population of Oscheius necromenus SB218 from Australian millipedes was also included in a phylogenetic comparison of what can now be characterized as a species complex of millipede-associated Oscheius. The only other nematode associates of millipedes belong to Rhigonematomorpha and Oxyuridomorpha, two strictly parasitic superorders of nematodes. These nematode identifications represent new geographic and host associations.

A novel cytochrome P450, CYP3201B1, is involved in (R)-mandelonitrile biosynthesis in a cyanogenic millipede.

Specialized arthropods and more than 2500 plant species biosynthesize hydroxynitriles and release hydrogen cyanide as a defensive mechanism. The millipede Chamberlinius hualienensis accumulates (R)‐mandelonitrile as a cyanide precursor. Although biosynthesis of hydroxynitriles in cyanogenic plants and in an insect are extensively studied, (R)‐mandelonitrile biosynthesis in cyanogenic millipedes has remained unclear. In this study, we identified the biosynthetic precursors of (R)‐mandelonitrile and an enzyme involved in (R)‐mandelonitrile biosynthesis. Using deuterium‐labelled compounds, we revealed that (E/Z)‐phenylacetaldoxime and phenylacetonitrile are the biosynthetic precursors of (R)‐mandelonitrile in the millipede as well as other cyanogenic organisms. To identify the enzymes involved in (R)‐mandelonitrile biosynthesis, 50 cDNAs encoding cytochrome P450s were cloned and coexpressed with yeast cytochrome P450 reductase in yeast, as cytochrome P450s are involved in the biosynthesis of hydroxynitriles in other cyanogenic organisms. Among the 50 cytochrome P450s from the millipede, CYP3201B1 produced (R)‐mandelonitrile from phenylacetonitrile but not from (E/Z)‐phenylacetaldoxime, whereas plant and insect cytochrome P450s catalysed the dehydration of aldoximes and hydroxylation of nitriles. CYP3201B1 is not phylogenetically related to cytochrome P450s from other cyanogenic organisms, indicating that hydroxynitrile biosynthetic cytochrome P450s have independently evolved in distant species. Our study will shed light on the evolution of cyanogenesis among plants, insects and millipedes.DatabaseNucleotide sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers LC125356–LC125405.

A sacrificial millipede altruistically protects its swarm using a drone blood enzyme, mandelonitrile oxidase.

Soldiers of some eusocial insects exhibit an altruistic self-destructive defense behavior in emergency situations when attacked by large enemies. The swarm-forming invasive millipede, Chamberlinius hualienensis, which is not classified as eusocial animal, exudes irritant chemicals such as benzoyl cyanide as a defensive secretion. Although it has been thought that this defensive chemical was converted from mandelonitrile, identification of the biocatalyst has remained unidentified for 40 years. Here, we identify the novel blood enzyme, mandelonitrile oxidase (ChuaMOX), which stoichiometrically catalyzes oxygen consumption and synthesis of benzoyl cyanide and hydrogen peroxide from mandelonitrile. Interestingly the enzymatic activity is suppressed at a blood pH of 7, and the enzyme is segregated by membranes of defensive sacs from mandelonitrile which has a pH of 4.6, the optimum pH for ChuaMOX activity. In addition, strong body muscle contractions are necessary for de novo synthesis of benzoyl cyanide. We propose that, to protect its swarm, the sacrificial millipede also applies a self-destructive defense strategy—the endogenous rupturing of the defensive sacs to mix ChuaMOX and mandelonitrile at an optimum pH. Further study of defensive systems in primitive arthropods will pave the way to elucidate the evolution of altruistic defenses in the animal kingdom.

Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede, Chamberlinius hualienensis

Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohydrins and causes the release of hydrogen cyanide (cyanogenesis). HNL can enantioselectively produce cyanohydrins, which are valuable building blocks for the synthesis of fine chemicals and pharmaceuticals, and is used as an important biocatalyst in industrial biotechnology. Currently, HNLs are isolated from plants and bacteria. Because industrial biotechnology requires more efficient and stable enzymes for sustainable development, we must continuously explore other potential enzyme sources for the desired HNLs. Despite the abundance of cyanogenic millipedes in the world, there has been no precise study of the HNLs from these arthropods. Here we report the isolation of HNL from the cyanide-emitting invasive millipede Chamberlinius hualienensis, along with its molecular properties and application in biocatalysis. The purified enzyme displays a very high specific activity in the synthesis of mandelonitrile. It is a glycosylated homodimer protein and shows no apparent sequence identity or homology with proteins in the known databases. It shows biocatalytic activity for the condensation of various aromatic aldehydes with potassium cyanide to produce cyanohydrins and has high stability over a wide range of temperatures and pH values. It catalyzes the synthesis of (R)-mandelonitrile from benzaldehyde with a 99% enantiomeric excess, without using any organic solvents. Arthropod fauna comprise 80% of terrestrial animals. We propose that these animals can be valuable resources for exploring not only HNLs but also diverse, efficient, and stable biocatalysts in industrial biotechnology.

A revision of the millipede genus Riukiupeltis Verhoeff, 1939 (Diplopoda, Polydesmida, Paradoxosomatidae), with comments on the status of related species

The East Asian millipede genus Riukiupeltis Verhoeff, 1939 is revised, and is restricted to a single species, Riukiupeltis jamashinai Verhoeff, 1939. Examination of the type specimens and freshly collected material from the Ryukyu Archipelago and Vietnam show that both subsequently allocated species, Riukiupeltis uenoi Murakami, 1975, and Riukiupeltis falcatus (originally Haplogonosoma falcatum Attems, 1953, reallocated by Jeekel 1968), do not belong to this genus; moreover, they are not even congeneric with each other. According to our morphological observations, including the gonopods, Riukiupeltis uenoi is closer to the widespread Chamberlinius hualienensis Wang, 1956, hence we propose the new combination Chamberlinius uenoi (Murakami, 1975), comb. n. Riukiupeltis falcatus, on the other hand, represents a separate, as yet monotypic, genus Simplogonomorpha gen. n., distinct both from Haplogonosoma Brölemann, 1916 sensu Golovatch et al. (1995), and from Verhoeff’s original Riukiupeltis. Additionally, Simplogonomorpha falcata (Attems, 1953), comb. n is re-described here based on fresh material from Vietnam. A key and colour habitus-illustrations to all three species are also provided here.

Revision of the Taiwanese millipede genus Chamberlinius Wang, 1956, with descriptions of two new species and a reclassification of the tribe Chamberlinini (Diplopoda, Polydesmida, Paradoxosomatidae, Paradoxosomatinae)

The millipede genus Chamberlinius is basically confined to Taiwan, with only one of the four known species presumably introduced to southern Japan. Both previously known species are redescribed, based on new material: Chamberlinius hualienensis Wang, 1956 (the type species) and Chamberlinius piceofasciatus (Gressitt, 1941), the latter being a new subjective senior synonym of Chamberlinius shengmui Wang, 1957, syn. n. Two further congeners are described as new: Chamberlinius pessior sp. n. and Chamberlinius sublaevus sp. n. The genus is re-diagnosed, all of its four species are keyed, and their distributions mapped. The tribe Chamberlinini is reclassified and, based on gonopod traits, shown to comprise the following five genera: Chamberlinius Wang, 1956, Haplogonosoma Brölemann, 1916, Riukiupeltis Verhoeff, 1939, Aponedyopus Verhoeff, 1939 and Geniculodesmus Chen, Golovatch and Chang, 2008.

Internal elements of the millipede, Chamberlinius hualienensis Wang (Polydesmida: Paradoxosomatidae)

The concentrations of 54 elements of the millipede Chamberlinius hualienensis and the soil including leaf litters were examined using inductively coupled plasma atomic emission spectrometry (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS). In all the millipedes, the Ca concentration derived from the calcium carbonate in the exoskeleton was the highest, at an average of 76 μg/mg-weight, and the other major elements were the following: Mg, K, Na, Fe, Al, Cu, Zn, Sr, Ba, Mn and Ti (>1 ng/mg-weight), whereas Ni, Cr, V, Ga, Mo, Co, Cd, etc. were at trace levels. Interestingly, the contents of Ca, Mg, K, Na, Zn, Sr, V, Co, Ag, and Sc in larvae were higher than in adult millipedes. Although the heavy metal contents, in the order of Cu>Pb>Cd, were similar to those of other invertebrates such as chilopoda, collembola, and carabidae, there was a high concentration of Cu in the millipedes. The other internal metals, As and Ag, were dependent on the soil concentrations in the habitat. Zn concentrations at sampling sites were different, but a constant level was maintained in the millipede. The results suggest that millipedes may provide information on the accumulation or regulation mechanism in the body for certain hazardous heavy metals. The C1 chondrite normalization pattern for lanthanoid series elements in both the millipede and soil indicated that the environmental habitats were well protected from pollution. The characteristics of internal elements and metal accumulation in the millipede in terms of relation to the habitat would be useful information for environmental pollution studies.

Secretions of Chamberlinius hualienensis Wang (Polydesmida: Paradoxosomatidae) during the reproductive migration stage.

Six minor secretory compounds: phenol, p-cresol, guaiacol, methyl benzoate, benzaldehyde dimethyl acetal and creosol have been identified from a methanol extract of the millipede Chamberlinius hualienensis Wang at the 7th nymphal and adult stages of reproductive migration. These compounds, except for methyl benzoate, were detected for the first time from C. hualienensis. Almost all female nymphs contained greater amounts of the compounds than male nymphs. In females the amounts of methyl benzoate and guaiacol remarkably decreased with development of the nymphal stage. Although only a defensive role has been postulated for millipede secretions, such a quantitative change in secretory compounds at the reproductive migration stage suggests they may possibly have a significant physiological function related to reproduction and/or development.