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.

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.

A new species of Ceratophysella (Collembola: Hypogastruridae) from Japan, with notes on its DNA barcode and a key to Japanese species in the genus.

Ceratophysella comosa sp. nov. was collected from ascomata of Ciborinia canielliae in Japan and the morphological and molecular characteristics of the species are described here. The species has 3 + 3 cephalic spines as in Ceratophysella loricata and Ceratophysella pilosa, but a plurichaetosis intermediate between C. loricata (absent) and C. pilosa (strong). The new species can be distinguished from these two species also by the number of setae on the first thorax segment and ventral tube. Partial DNA sequences of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene were used as DNA barcodes to distinguish species. Interspecific genetic distances of the gene were higher than the intraspecific distances between Ceratophysella species for which sequence data are available. An identification key of Japanese Ceratophysella is provided.

Contribution to the taxonomy of Lobellini (Collembola: Neanurinae) and investigations on luminous Collembola from Japan.

Lobella sauteri was redescribed based on the lectotype and specimens obtained from the type locality Bugenji, Yokohama, Kanagawa, as the true identity of the luminous Collembola, Lobella sp. Lobella sauteri has morphological traits characteristic of the genus currently called Telobella. As L. sauteri is the type species of Lobella, the genus Telobella was synonymised with Lobella according to the principle of priority, and the genus Lobella was redefined to include both the species previously assigned to Telobella and those previously assigned to Lobella. A new species Lobella monstrum sp. nov. was described and new combinations were proposed for certain species in Lobellini. Light-emitting capacity was confirmed in L. sauteri and newly reported in Lobella yambaru comb. nov. Vitronura giselae and Vitronura kunigamiensis.

A new Anurida species of the hammerae-group from the Russian Far East (Collembola, Neanuridae).

A new species of the genus Anurida is described and illustrated based on material from the Khabarovsk Territory, Russia: A. hirsuta sp. nov. This new species belongs to the amphi-Pacific hammerae-group and it seems to be particularly similar to the Beringian A. hammerae Christiansen, 1952 and the Japanese A. trioculata Kinoshita, 1916. Anurida hirsuta sp. nov. can easily be distinguished from both latter species primarily by its peculiar dorsal chaetotaxy, apparently the most complete among all congeners. A key is presented to all eight species presently known to compose the group.

A review of Pseudachorutes Tullberg, 1871 (Collembola, Neanuridae) from the East Asia, with description of six new species.

A review of the Pseudachorutes fauna of the East Asia was performed, mainly based on fresh material from forest ecosystems of the Russian Far East and some adjoining regions. Six new species of the genus, namely P. aleksandrae sp. nov., P. armatus sp. nov., P. minimus sp. nov., P. variabilis sp. nov., P. concinnus sp. nov. and P. morulifer sp. nov., are described and extensive remarks on further six congeners, i.e. P. andrei Weiner Najt, P. hitakamiensis Tamura, P. isawaensis Tamura, P. kangchenjungae Yosii, P. longisetis Yosii and P. polychaetosus Gao Palacios-Vargas, previously known from the studied region, are given. Pseudachorutes conicus Lee Kim and P. sibiricus Rusek, are considered as probable junior synonyms of P. longisetis and P. andrei, respectively. Pseudachorutes boerneri SchÓ§tt is recorded for the first time in the eastern Palaearctic and redescribed based on materials from Yakutia, Khabarovsk Territory and Southern Primor'e. A regional key to the mentioned species and a summarizing table of diagnostic characters of all regional congeners are also provided.

Genome-wide gene expression profiling in gamma-irradiated green alga, Pseudokirchneriella subcapitata, by HiCEP.

Transcriptome was analyzed in gamma-irradiated green alga Pseudokirchneriella subcapitata by high coverage gene expression profiling (HiCEP). Approximately 7,800 expressed genes were detected. Expression levels of 623-707 genes were affected at 100-300 Gy. Nucleotide sequences of 41 up-regulated genes were determined. The quantitative reverse transcription polymerase chain reaction validated the up-regulation. Two genes had homology to genes related to ionizing radiation. These results indicate usefulness of HiCEP for screening of stress-responsive genes in species that are ecotoxicologically important but for which genomic sequence information is lacking.

Model construction for estimating potential vulnerability of Japanese soils to cadmium pollution based on intact soil properties.

Prediction of heavy metal bioavailability in intact soil is important to manage soil pollution risks. We developed a regression model for representative Japanese soils to judge their potential vulnerability to cadmium (Cd) pollution. We added four rates of Cd to 17 sample soils to mimic artificial contamination. After aging the contaminated soils, we measured Cd's bioavailability using the diffusive gradients in thin-films (DGT) technique. We then evaluated the relationships between bioavailability of Cd ([CdDGT]) and intact soil properties by statistical analyses. Cation exchange capacity (CEC) and pH emerged as significant factors to explain the cadmium bioavailability in Japanese soils. Specifically, lower CEC and lower pH were associated with higher [CdDGT], which poses a higher risk for soil ecosystems. The correlation between pH and [CdDGT] had a high dependence on [CdAdd], whereas that for CEC did not. Regression analysis also showed that the interaction between intact soil pH and spiked concentration ([CdAdd]) had a significant contribution to [CdDGT]. The regression model developed was rationally supported by a biotic ligand model. This simplified but realistic model would be useful in estimating the vulnerability of representative Japanese soils and determining the risk for Japanese soils in relation to Cd contamination.

Effects of acute gamma irradiation on Folsomia candida (Collembola) in a standard test.

An understanding of the effects of ionizing radiation on non-human biota is required by the International Commission on Radiological Protection for the radiological protection of the environment. We examined dose-effect relationships for gamma radiation on survival, growth, and reproduction in the soil invertebrate Folsomia candida (Collembola) in a standard laboratory test. F. candida were acutely irradiated at increasing doses of gamma radiation, and subsequent survival, growth in body length, and number of neonates produced by irradiated specimens were examined. The 50% lethal dose was at 1356 Gy, and the 10% and 50% effective doses (ED10 and ED50) for growth were at 32 and 144 Gy, respectively. The ED10 and ED50 values for reproduction were at 7.1 and 21.9 Gy, respectively. These data establish important baselines for the radiological protection of terrestrial ecosystems based on scientific principles.

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.

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.

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 (baker's 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.

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.

mRNA expression of a cadmium-responsive gene is a sensitive biomarker of cadmium exposure in the soil collembolan Folsomia candida.

The gene expression of environmental organisms is useful as a biomarker of environmental pollution. One of its advantages is high sensitivity. We identified the cDNA of a novel cadmium-responsive gene in the soil collembolan Folsomia candida. The deduced protein, designated "metallothionein-like motif containing protein" (MTC), was cysteine-rich and contained a metallothionein-like motif with similarity to metallothionein, but had a much longer sequence than metallothionein and contained repeated sequences of amino acids. Expression of MTC mRNA was sensitively induced by cadmium exposure at 0.3 mg/kg of dry food, a concentration at which toxic effects are not observed, but expression was not affected by gamma-ray exposure (an inducer of oxidative stress). These findings suggest that MTC is involved in cadmium-binding processes rather than in oxidative-stress responses. In conclusion, we suggest that gene expression of MTC may be a candidate biomarker for detecting low levels of cadmium contamination in soil.

Application of HiCEP to screening of radiation stress-responsive genes in the soil microarthropod Folsomia candida (Collembola).

The field of ecotoxicogenomics has received increasing attention for its potential to provide insight into pressing ecological issues. However, its applications are limited due to a lack of genetic sequence information for organisms used in ecotoxicological studies. We used high-coverage expression profiling (HiCEP), a method that requires no prior sequence knowledge, to examine stress-responsive genes and their dose dependence in the springtail Folsomia candida using gamma radiation as the stressor. Radiation-responsive genes and their dose dependency were detected at effective doses for reproduction, and 16 up-regulated transcript-derived fragments (TDFs) were sequenced. Quantitative PCR analysis also found that most of the TDFs were up-regulated. The sequences of the TDFs showed resemblance to known genes, such as glutathione S-transferase and poly(ADP-ribose) polymerase, but most showed no similarity to any genes in the gene databases. These results suggest that HiCEP is effective for discovering differently expressed genes and their dose dependence, even in organisms for which few sequence data are available. The limited length of the TDFs, however, may impede functional annotation of the genes. In conclusion, HiCEP is useful for ecotoxicogenomic studies in which various organisms with few available genomic resources are involved.

Defensive role of cystidia against Collembola in the basidiomycetes Russula bella and Strobilurus ohshimae.

Cystidia in fruit bodies are taxonomically important characters. However, little is known about their ecological functions. The defensive role of cystidia against the collembolans Ceratophysella denisana and Mitchellania horrida was examined in fruit bodies of Russula bella and Strobilurus ohshimae. Cystidium-destruction experiments demonstrated that R. bella and S. ohshimae cystidia decrease the number of collembola found on gills, although the effects were not significant for R. bella against C. denisana. Furthermore, R. bella cystidia increased collembolan mortality in the laboratory, and in the field, collembola were found dead on parts of the fruit body of this species where cystidia were abundant. In the cystidium-destruction experiment, approximately one-third of collembola appeared to avoid R. bella. Therefore, deadly cystidia may be selected for in R. bella to avoid collembolan attack. Laboratory feeding experiments revealed that collembola can extensively damage R. bella and S. ohshimae basidiospores by feeding. These results suggest that the cystidia of R. bella and S. ohshimae may protect basidiospores from collembolan predation.