Satoshi Shimano

Scheloribatid Mites as the Source of Pumiliotoxins in Dendrobatid Frogs

The strawberry poison frog Dendrobates pumilio (Anura: Dendrobatidae) and related poison frogs contain a variety of dendrobatid alkaloids that are considered to be sequestered through the consumption of alkaloid-containing arthropods microsympatrically distributed in the habitat. In addition to ants, beetles, and millipedes, we found that adults of two species of oribatid mites belonging to the cohort Brachypylina, trophically a lower level of animal than ants and beetles, contain dendrobatid alkaloids. Gas chromatography/mass spectrometry (GC/MS) of hexane extracts of adult Scheloribates azumaensis (Oribatida: Acari) revealed the presence of not only pumiliotoxin 251D (8-hydroxy-8-methyl-6-(2′-methylhexylidene)-1-azabicyclo[4.3.0]nonane), but also precoccinelline 193C and another coccinelline-type alkaloid. From the corresponding extracts of an unidentified Scheloribates sp., pumiliotoxin 237A (8-hydroxy-8-methyl-6-(2′-methylpentylidene)-1-azabicyclo[4.3.0]nonane) was detected as a minor component, and identified by synthesis. The presence of related alkaloids, namely deoxypumiliotoxin 193H, a 6,8-diethyl-5-propenylindolizidine, and tentatively, a 1-ethyl-4-pentenynylquinolizidine, were indicated by the GC/MS fragmentation patterns, along with at least another six unidentified alkaloid components. Thus, one possible origin of pumiliotoxins, coccinellid alkaloids, and certain izidines found in poison frogs may be mites of the genus Scheloribates and perhaps related genera in the suborder Oribatida.

Morphological and Molecular Phylogeny of Dileptid and Tracheliid Ciliates: Resolution at the Base of the Class Litostomatea (Ciliophora, Rhynchostomatia)

Dileptid and tracheliid ciliates have been traditionally classified within the subclass Haptoria of the class Litostomatea. However, their phylogenetic position among haptorians has been controversial and indicated that they may play a key role in understanding litostomatean evolution. In order to reconstruct the evolutionary history of dileptids and tracheliids, and to unravel their affinity to other haptorians, we have used a cladistic approach based on morphological evidence and a phylogenetic approach based on 18S rRNA gene sequences, including eight new ones. The molecular trees demonstrate that dileptids and tracheliids represent a separate subclass, Rhynchostomatia, that is sister to the subclasses Haptoria and Trichostomatia. The Rhynchostomatia are characterized by a ventrally located oral opening at the base of a proboscis that carries a complex oral ciliature. We have recognized two orders within Rhynchostomatia. The new order Tracheliida is monotypic, while the order Dileptida comprises two families: the new, typically bimacronucleate family Dimacrocaryonidae and the multimacronucleate family Dileptidae. The Haptoria evolved from the last common ancestor of the Litostomatea by polarization of the body, the oral opening locating more or less apically and the oral ciliature simplifying. The Trichostomatia originated from a microaerophylic haptorian by further simplification of the oral ciliature, possibly due to an endosymbiotic lifestyle.

Chemical ecology of oribatid mites III. Chemical composition of oil gland exudates from two oribatid mites, Trhypochthoniellus sp. and Trhypochthonius japonicus (Acari: Trhypochthoniidae).

The composition of oil gland exudates from two oribatid mites, Trhypochthoniellus sp. and Trhypochthoniusjaponicus, was studied with reference to the related species Trhypochthonielluscrassus. Trhypochthoniellus sp. contained a mixture of seven compounds; (Z,Z)-6,9-heptadecadiene, geranial, 3-hydroxybenzene-1,2-dicarbaldehyde (γ-acaridial), neryl formate, neral, (Z)-8-heptadecene and geranyl formate in decreasing order of abundance. The profile of the components from T. japonicus consisted of two types depending on the locality of sampling with unknown reason; one possessing a mixture of eight compounds [(Z,E)-farnesal, γ-acaridial, (Z,Z)-6,9-heptadecadiene, (E,E)-farnesal, (Z)-8-heptadecene and geranial in decreasing order] together with two unknown compounds, and the other composed of the same set of compounds together with 2-hydroxy-6-methylbenzaldehyde as the most abundant component. Relative abundance among common components was consistent between the two types of T. japonicus. Profiles of components differed among three species including T. crasus. The phylogenetic relationship between Oribatida and Astigmata was discussed based on secretory compounds commonly distributed between these two suborders.

Morphology and Gene Sequence of Levicoleps biwae n. gen., n. sp. (Ciliophora, Prostomatida), a Proposed Endemic from the Ancient Lake Biwa, Japan

ABSTRACT. Levicoleps biwae n. gen., n. sp. was discovered in organic mud on the shore of Lake Biwa, Japan. Its morphology and small subunit rRNA gene sequence were studied with standard methods. Further, we established a terminology for the colepid armour and selected four features for genus recognition: the number of armour tiers, the structure of the tier plates, the presence/absence of armour spines, and the number of adoral organelles (three or five). The Japanese colepid, a barrel‐shaped ciliate with an average size of 75 × 45 μm, has six armour tiers and hirtus‐type tier plates, but lacks armour spines, both in the environment and in laboratory culture. Thus, it is considered to represent a new genus. This rank is supported by the considerable genetic distance (7%) from the common Coleps hirtus. Although L. biwae looks quite similar to C. hirtus in vivo, it is very likely most closely related to Coleps amphacanthus, a species with conspicuous armour spines, as indicated by body size, the number of ciliary rows and, especially, the multiple caudal cilia. Lake Biwa is about four million years old and inhabited by many endemic organisms, ranging from algae to large fish. Thus, we suspect that L. biwae is restricted to Lake Biwa or, at least, to Asia. Based on literature data and the generic features established, we also propose the new genus Reticoleps for Coleps remanei Kahl, 1933, and resurrect the genus Pinacocoleps Diesing, 1865 to include Coleps incurvus Ehrenberg, 1833, Coleps pulcher Spiegel, 1926, Coleps tessalatus Kahl, 1930 and, probably, Baikalocoleps quadratus Obolkina, 1995a. Nine colepid genera are diagnosed and dichotomously keyed.

Ophiocordyceps pulvinata sp. nov., a pathogen with a reduced stroma

Ophiocordyceps pulvinata, a pathogen of ants, is formally described as a new species. Genus level designation of this species is difficult due to several apparently conflicting morphological and ecological characters. Affinity with Ophiocordyceps is suggested by the dark color stroma and ascospore morphology. However, the species was included in a book of entomopathogenic fungi of Japan as Torrubiella sp. due to the production of perithecia on an astipitate stroma. Phylogenetic analyses of molecular data support a close relationship with O. unilateralis, a finding consistent with morphological characteristics of the color, asci and ascospores and ecological traits of host affiliation. Thus, O. pulvinata represents another example of the loss of stipe for the hypocrealean arthropod pathogenic fungi and highlights the utility of asci and ascospore morphology as taxonomically informative characters of closely related taxa.

Morphological and molecular characterization of Histiobalantium natans viridis (Ciliophora, Scuticociliatia)

We investigated a Histiobalantium natans viridis population from the ancient Lake Biwa in Japan, using live observation, silver impregnation, and the small subunit rRNA gene sequence. The morphological and molecular data show, with high support, a close relationship of Histiobalantium, Schizocalyptra and Pleuronema, supporting the family Pleuronematidae Kent, whose nearest relatives are the Cyclidiidae Ehrenberg. A family Histiobalantiidae Puytorac and Corliss is not supported, either by the nucleotide sequences or the morphologic data, except for the curious dorsal location of the cytopyge. Likewise, the data do not support inclusion of Histiobalantium in the family Sulcigeridae Gajewskaja, as very recently suggested by Jankowski, whose classification is based on a misidentified Sulcigera comosa. Further, there are good reasons to synonymize the genus Gajewskiella Obolkina with Histiobalantium. The European and Asian populations of H. natans differ significantly in the structure of adoral membranelle 1, suggesting that they are different species. However, there is some indication that the differences are caused by deficient data on the European H. natans, which is thus in need of detailed redescription. This applies also to the North American Histiobalantium agile Stokes which is probably not conspecific with the Eurasian H. natans. The status of the forma H. natans viridis and H. natans nigricans is not known because detailed data from nigricans are lacking. We prefer subspecific rank at the present state of knowledge.

Multicolor staining of root systems in pot culture

Abstract We have developed a method for staining the root systems of neighboring plants distinguishably in pot culture to facilitate studies of the interactions between plants. Pot soil was desiccated until the plant wilted, and then the shoot was cut and a dye solution (Fantasy) was pressure-injected into the roots at 0.05 MPa (gauge). All the roots, including fine roots of double-planted tomato (Lycopersicon esculentum var. Momotaro), in a 10-cm-diameter pot were well stained, but the root hairs were not. The volume of dye and the time required for staining were 6.4 mL and 5 h, respectively (root length 179 m plant−1; root fresh weight 2.62 g plant−1 on average). The root distribution of double-planted tomato in a 40 L container was well determined. The volume of dye and the time required for staining were 100 mL and 40 h, respectively (root length 1,514 m plant−1; root dry weight 4.93 g plant−1 on average). Each plants roots accounted for 37–52% of the total root weight at an intermediate position between the two plants, whereas at the other positions, the roots of either plant were dominant (82–99% of total root weight). In principle, the proposed method is applicable to whole plant species with vascular systems.

Diversity of Fungi on Decomposing Leaf Litter in a Sugarcane Plantation and Their Response to Tillage Practice and Bagasse Mulching: Implications for Management Effects on Litter Decomposition

To minimize the degradation of soil organic matter (SOM) content in conventional sugarcane cropping, it is important to understand how the fungal community contributes to SOM dynamics during the decomposition of sugarcane leaf litter. However, our knowledge of fungal diversity in tropical agroecosystems is currently limited. Thus, we determined the fungal community structure on decomposing sugarcane leaf litter and their response to different soil management systems using the internal transcribed spacer region 1 (ITS1) amplicon sequencing method afforded by Ion Torrent Personal Genome Machine (PGM). The results indicate that no-tillage had positive effects on the relative abundance of Zygomycota and of some taxa that may prefer a moist environment over conventional tillage, whereas bagasse mulching decreased the richness of operational taxonomic units (OTUs) and had positive effect on the relative abundance of slow-growing taxa, which may prefer poor nutrient substrates. Furthermore, a combination of no-tillage and bagasse mulching increased the abundance of unique OTUs. We suggest that the alteration of fungal communities through the changes in soil management practices produces an effect on litter decomposition.

Shifts in the composition and potential functions of soil microbial communities responding to a no-tillage practice and bagasse mulching on a sugarcane plantation

This study examined the effect of conversion from a conventional tillage (CT) to a no-tillage (NT) practice and of bagasse mulching (M) on soil microbial community composition and potential functions, using phospholipid fatty acid (PLFA) analysis and shotgun metagenome sequencing. Our results showed that both the NT and the M treatment increased microbial PLFAs. The shotgun sequencing results suggested that the functional profiles are more resistant to agricultural managements than to community compositions, which supports the hypothesis of the functional redundancy of soil microbial communities. However, some metabolism-related sequences were significantly affected by different treatments. The percentage of sequences related to metabolism of carbohydrates, especially saccharide groups, was significantly higher in the CT soils than in NT and M soils, which may be linked to lower carbon (C) availability in CT soils. Compared with CT, the NT had higher alpha diversity and more sequences related to DNA metabolism, which may be associated with higher nutrient availability. On the other hand, the M treatment decreased the percentages of sequences related to the metabolism of amino acids and derivatives, which may be due to the limited nitrogen (N) because of the high C/N ratio of bagasse. We also observed interaction effects of the NT and M treatments; although both the NT and M treatments increased the relative abundance of Proteobacteria, this variable in NT + M soils was not higher than in each single treatment. Overall, our findings suggest that the microbial communities change their composition and functionality in response to the NT and M treatments, and these shifts have the potential to affect important soil processes that sustain crop productivity, such as C sequestration and major nutrient cycles.

Improvement of root staining method for field applications

The root interaction between neighboring plants is neither obvious nor well studied because there are no easy and convenient methods to distinguish the root system among individual plants. Previously, we developed a method of pressure–injection of dye into the roots of pot-grown plants in dry soil condition. Here, we modified the method for high-pressure injection in wet soil or outdoor fields to counter root pressure. In our preliminary pot experiment, the roots of tomato plants (Lycopersicon esculentum “Momotaro”) were stained well in wet soil by dye injection at 0.5 MPa. In the field test, tomato plants were grown in a 2 m × 2 m concrete frame field. Four successive plant shoots were cut 159 and 170 days after sowing, and dye solutions (Fantasy) of different colors were pressure-injected into the roots from the stumps from 0.05 to 0.5 MPa. The volume of the dye and the time required for staining were respectively in the ranges of 120 to 150 mL and 9 to 96 h. Soil blocks or cores were sampled and the roots were collected by hand picking and the water floating method, and distinguished by color. The recognition and collection of roots were easy because most of them were stained dark. In the 0–15 cm soil layer, most of the roots in the four blocks surrounding the hills came from the hills. In blocks found at the intermediate position between two adjacent plants, the roots of both plants were distributed. In the 15–30 cm soil layer, most of the roots in the four blocks surrounding the hills came from the hills, although roots of other plants also existed. The roots of each plant extended to adjacent plants, even reaching distant plants. The roots of a plant did not grow toward another plant in the opposite row. Findings of this study are useful in relation to effective fertilizer application. The proposed injection method is expected to facilitate the study of the interactions between root systems in the field.