Taizo Nakamori

Radiocesium immobilization to leaf litter by fungi during first-year decomposition in a deciduous forest in Fukushima.

Vast forest areas in eastern Japan have been contaminated with radio-isotopes by the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident. Radiocesium (radioCs) is known to remain bioavailable in forest ecosystems for a long time, and it is necessary to terminate the cycling process to decontaminate the forest ecosystem. We observed radiocesium concentrations of leaf litter during decomposition on a forest floor where radiocesium ((137)Cs) contamination was ∼155 kBq/m(2). Litter bag experiments were conducted with newly fallen mixed deciduous leaf litter in a deciduous forest (alt. 610 m) about 50 km from the FDNPP. Litter bags were retrieved in April, June, August, October, and December 2012. Fresh litter (137)Cs concentration was ∼3000 Bq/kg in December 2011. During the decomposition process on the forest floor, litter (137)Cs concentration increased rapidly and exceeded 25,000 Bq/kg after 6 months, whereas potassium (K) concentration in the litter was rather stable, indicating that radiocesium and K showed contrasting dynamics during the early decomposition phase. Nitrogen, phosphorus, and (137)Cs contents were positively correlated to fungal biomass, evaluated by phospholipid fatty acids in the litter during decomposition. The increase of radiocesium concentration mainly occurred during from April to October, when fungal growth peaked. Therefore, this suggests fungal translocation of nutrients from outside the litter substrate (immobilization) is the mechanism to increase radiocesium in the decomposing litter. The amount of (137)Cs contained in the 1-year-old decomposed leaf litter was estimated to be 4% per area of the soil-contaminated (137)Cs.

A quantitative protocol for DNA metabarcoding of springtails (Collembola).

We developed a novel protocol with superior quantitative analysis results for DNA metabarcoding of Collembola, a major soil microarthropod order. Degenerate PCR primers were designed for conserved regions in the mitochondrial cytochrome c oxidase subunit I (mtCOI) and 16S ribosomal RNA (mt16S) genes based on published collembolan mitogenomes. The best primer pair was selected based on its ability to amplify each gene, irrespective of the species. DNA was extracted from 10 natural communities sampled in a temperate forest (with typically 25-30 collembolan species per 10 soil samples) and 10 mock communities (with seven cultured collembolan species). The two gene regions were then amplified using the selected primers, ligated with adapters for 454 technology, and sequenced. Examination of the natural community samples showed that 32 and 36 operational taxonomic units (defined at a 90% sequence similarity threshold) were recovered from the mtCOI and mt16S data, respectively, which were comparable to the results of the microscopic identification of 25 morphospecies. Further, sequence abundances for each collembolan species from the mtCOI and mt16S data of the mock communities, after normalization by using a species as the internal control, showed good correlation with the number of individuals in the samples (R = 0.91-0.99), although relative species abundances within a mock community sample estimated from sequences were skewed from community composition in terms of the number of individuals or biomass of the species. Thus, this protocol enables the comparison of collembolan communities in a quantitative manner by metabarcoding.

Biomarker responses reveal that food quality affects cadmium exposure in the soil collembolan Folsomia candida

Food quality affects the food consumption rate, flux through the gut, and exposure to contaminants in animals. This study evaluated the effects of food quality on cadmium exposure in the soil collembolan Folsomia candida. Animals were exposed to constant concentrations of cadmium for 38 h via artificial food consisting of an agar medium with various concentrations of sugar (glucose), total nutrients (bakers yeast), or fungal odour (1-octen-3-ol). The expression of the gene encoding a deduced metallothionein-like motif containing protein was used as a biomarker of cadmium exposure. Glucose concentrations of 2% or higher reduced the expression levels of the biomarker. Within the range of 0.1-8% yeast, medium concentrations led to higher biomarker levels. At high concentrations of 1-octen-3-ol (2000 mg/l), feeding and the biomarker response were reduced. These results suggest that even at equivalent cadmium concentrations, food quality affects cadmium exposure by altering food consumption rates.

Repellency of injured ascomata of Ciborinia camelliae and Spathularia flavida to fungivorous collembolans

The repellency of injured ascomata of Ciborinia camelliae and Spathularia flavida was tested with the collembolan Ceratophysella denisana, a common mushroom feeder. Presentation of a test ascoma (intact or injured) at a distance to a collembolan that was feeding at a bait showed that significantly more collembolans moved away in response to the presentation of injured ascomata than intact ones for both species. These results suggest that the ascomata of these species showed repellency to the collembolan species as a response to injury. Odor is speculated to be one of the stimuli eliciting the behavioral responses observed.

A rapid method of non-destructive DNA extraction from individual springtails (Collembola)

In this study, we describe an easy and rapid method for non-destructive DNA extraction from a single Collembola individual, without dissection, lysis of the specimen, or purification of extracted DNA. We demonstrate that, after a single specimen has been heat-treated in Tris-EDTA (TE) buffer using a standard thermocycler, the solution can be used for PCR amplification of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene region, typically used for DNA barcoding. With this method, the morphological features of Collembola commonly used for species identification are well preserved. This DNA extraction method is preferable for DNA barcoding where the sequencing and preservation of a large number of small and fragile specimens are required.

ENVIRONMENTAL PROTECTION: RESEARCHES IN NATIONAL INSTITUTE OF RADIOLOGICAL SCIENCES

Some studies for radiological protection of the environment have been made at the National Institute of Radiological Sciences (NIRS). Transfer of radionuclides and related elements has been investigated for dose estimation of non-human biota. A parameter database and radionuclide transfer models have been also developed for the Japanese environments. Dose (rate)-effect relationships for survival, growth and reproduction have been investigated in conifers, Arabidopsis, fungi, earthworms, springtails, algae, duckweeds, daphnia and medaka. Also genome-wide gene expression analysis has been carried out by high coverage expression profiling (HiCEP). Effects on aquatic microbial communities have been studied in experimental ecosystem models, i.e., microcosms. Some effects were detected at a dose rate of 1 Gy day(-1) and were likely to arise from interspecies interactions. The results obtained at NIRS have been used in development of frameworks for environmental protection by some international bodies, and will contribute to environmental protection in Japan and other Asian countries.

Biomass estimation of the terrestrial ecotoxicological species Folsomia candida (Collembola) using a real-time polymerase chain reaction

The abundance and growth of the Folsomia candida soil arthropod have been widely used to assess the environmental impact of a range of soil pollutants, and increasing concerns about environmental pollution require advanced and rapid methods to estimate ecological toxicity. Here, we developed a quantitative polymerase chain reaction (qPCR)-based assay for determining the biomass of F. candida. Prior to DNA extraction, an appropriate amount of an artificial sequence was spiked into the test samples, allowing us to assess the extraction efficiency used for normalisation. We designed primers based on the sequencing information of the nuclear RNA polymerase II (Pol II) and mitochondrial cytochrome c oxidase subunit I (mtCOI) genes of F. candida. Assays were performed on samples containing a different number of individuals at the same body length (individually same biomass; same age) and samples containing the same number of individuals at a different body length (individually different biomass; different age). Biomass was estimated from the body lengths of collembolan samples. For both genes, DNA quantity showed a significant linear relationship between increased collembolan numbers and the estimated biomass; DNA quantity in different ages of collembolans showed a significant correlation with body length and a linear relationship with the estimated biomass. We believe that this rapid and accurate technique could be used to detect and quantify soil animals and thus would improve ecotoxicological testing.

Occurrence and gut contents of flatworms on fungal sporocarps

Terrestrial flatworms of the family Rhynchodemidae were collected from fungal sporocarps. The frequency of flatworms on sporocarps differed among fungal species. Gut content analysis showed that the flatworms preyed upon fungivorous Collembola or other arthropods on the sporocarps. Fungal materials were observed in flatworm guts but only on the body surface and in the gut of Collembola prey. Basidiospores were observed on the surfaces of the flatworms.

Two new littoral species of the genus Anurida Laboulbène, 1865 (Collembola, Neanuridae) from the Pacific coast of Asia

Two new littoral species of the genus Anurida, viz. A. kyshyensis sp. nov. and A. abashiriensis sp. nov., have been described from the eastern coasts of Chukchi Peninsula (Russia) and Hokkaido Island (Japan), respectively. The former species is most similar to sympatric A. similis Fjellberg, 1985 and A. martynovae Fjellberg, 1985 differing from both of them due to the permanent presence of additional lateral setae on thorax and 3+3 axial setae on Abd.4. A. abashiriensis sp. nov., having unique mandibles and hypertrophic elongate maxillae with long lamellae, is hard to compare with any known species of Anurida and allied genera.