Toll signalling promotes blastema cell proliferation during cricket leg regeneration via insect macrophages.

Hemimetabolous insects, such as the two-spotted cricket Gryllus bimaculatus, can recover lost tissues, in contrast to the limited regenerative abilities of human tissues. Following cricket leg amputation, the wound surface is covered by the wound epidermis, and plasmatocytes, which are insect macrophages, accumulate in the wound region. Here, we studied the function of Toll-related molecules identified by comparative RNA sequencing during leg regeneration. Of the 11 Toll genes in the Gryllus genome, expression of Toll2-1, Toll2-2 and Toll2-5 was upregulated during regeneration. RNA interference (RNAi) of Toll, Toll2-1, Toll2-2, Toll2-3 or Toll2-4 produced regeneration defects in more than 50% of crickets. RNAi of Toll2-2 led to a decrease in the ratio of S- and M-phase cells, reduced expression of JAK/STAT signalling genes, and reduced accumulation of plasmatocytes in the blastema. Depletion of plasmatocytes in crickets using clodronate also produced regeneration defects, as well as fewer proliferating cells in the regenerating legs. Plasmatocyte depletion also downregulated the expression of Toll and JAK/STAT signalling genes in the regenerating legs. These results suggest that Spz-Toll-related signalling in plasmatocytes promotes leg regeneration through blastema cell proliferation by regulating the Upd-JAK/STAT signalling pathway.

Anti-malarial activity in a Chinese herbal supplement containing Inonotus obliquus and Panax notoginseng.

Plasmodium falciparum, the most virulent human malaria parasite, causes serious diseases among the infected patients in the world and is particularly important in African regions. Although artemisinin combination therapy is recommended by the WHO for treatment of P. falciparum-malaria, the emergence of artemisinin-resistant parasites has become a serious issue which underscores the importance of sustained efforts to obtain novel chemotherapeutic agents against malaria. As a part of such efforts, thirty-nine herbal extracts from traditional Chinese medicine (TCM) were assayed for their anti-malarial activity using 3D7 strain of P. falciparum. Three herbal supplements appeared to possess higher specific anti-malarial activity than the others. One of them (D3) was separated by two sequential fractionations with reverse-phase (the first step) and normal-phase (the second step) liquid chromatography, in which some fractions resulted in higher specific activities than those of D3 or the previous fractions. Cell toxicity assay was performed with the fractions of the first fractionation and demonstrated no obvious cell toxicity. These results suggest that structure determination of the major compound for the anti-malarial activity in D3 may help the development of more potent chemicals in the future.

Evaluation of host effects on ectomycorrhizal fungal community compositions in a forested landscape in northern Japan.

Community compositions of ectomycorrhizal (ECM) fungi are similar within the same host taxa. However, careful interpretation is required to determine whether the combination of ECM fungi and plants is explained by the host preference for ECM fungi, or by the influence of neighbouring heterospecific hosts. In the present study, we aimed to evaluate the effects of host species on the ECM community compositions in a forested landscape (approx. 10 km) where monodominant forest stands of six ECM host species belonging to three families were patchily distributed. A total of 180 ECM operational taxonomic units (OTUs) were detected with DNA metabarcoding. Quantitative multivariate analyses revealed that the ECM community compositions were primarily structured by host species and families, regardless of the soil environments and spatial arrangements of the sampling plots. In addition, 38 ECM OTUs were only detected from particular host tree species. Furthermore, the neighbouring plots harboured similar fungal compositions, although the host species were different. The relative effect of the spatial factors on the ECM compositions was weaker than that of host species. Our results suggest that the host preference for ECM fungi is the primary determinant of ECM fungal compositions in the forested landscape.

Biogeographic Patterns of Ectomycorrhizal Fungal Communities Associated With Castanopsis sieboldii Across the Japanese Archipelago.

Biogeographic patterns in ectomycorrhizal (ECM) fungal communities and their drivers have been elucidated, including effects of host tree species and abiotic (climatic and edaphic) conditions. At these geographic scales, genotypic diversity and composition of single host tree species change with spatial and environmental gradients, reflecting their historical dispersal events. However, whether the host genotypes can be associated with the biogeographic patterns of ECM communities remains unclear. We investigated the biogeographic pattern of ECM fungal community associated with the single host species Castanopsis sieboldii (Fagaceae), whose genotypic diversity and composition across the Japanese archipelago has already been evaluated. ECM communities were investigated in 12 mature Castanopsis-dominated forests covering almost the entire distribution range of C. sieboldii, and we quantified the effect of host genotypes on the biogeographic pattern of ECM fungal communities. Richness and community composition of ECM fungi changed with latitude and longitude; these biogeographic changes of ECM community were significantly correlated with host genotypic variables. Quantitative analyses showed a higher relative explanatory power of climatic and spatial variables than that of host genotypic variables for the biogeographic patterns in the ECM community. Our results suggest historical events of host dispersal can affect the biogeographic patterns of the ECM fungal community, while their explanation power was lower than that for climatic filtering and/or fungal dispersal.

Temporal distance decay of similarity of ectomycorrhizal fungal community composition in a subtropical evergreen forest in Japan.

Community compositions of ectomycorrhizal (ECM) fungi are known to show spatial distance decay of similarity, which arises from both deterministic niche-based processes and stochastic spatial-based processes (e.g. dispersal limitation). Recent studies have highlighted the importance of incorporating the spatial-based processes in the study of community ecology of ECM fungi. However, few studies have investigated the temporal distance decay of similarity of ECM fungal communities. More specifically, the role of stochastic temporal-based processes, which could drive the temporal distance decay of similarity independently of niche-based processes, in the temporal variation of the communities remains unclear. Here we investigated ECM fungi associated with roots of Castanopsis sieboldii at 3-month intervals over a 2-year period. We found that dissimilarity of the ECM fungal community composition was significantly correlated with temporal distance but not with environmental distance among sampling dates. Both climatic and temporal variables significantly explained the temporal variation of the community composition. These results suggest that temporal variations of ECM fungi can be affected not only by niche-based processes but also by temporal-based processes. Our findings imply that priority effects may play important roles in the temporal turnover of ECM fungal community at the site.

Disentangling the relative importance of host tree community, abiotic environment and spatial factors on ectomycorrhizal fungal assemblages along an elevation gradient.

Recent studies have shown that changes in community compositions of ectomycorrhizal (ECM) fungi along elevation gradients are mainly affected by changes in host tree communities and/or in abiotic environments. However, few studies have taken the effects of processes related to fungal dispersal (i.e. spatial processes) into account and distinguished the effects of host community, abiotic environment and spatial processes on community composition along elevation gradients. This has left unclear the relative importance of these factors in structuring the ECM community assemblages. To address this, we investigated the community composition of ECM fungi along an elevation gradient in northern Japan with 454 meta-barcoding. We found that the community composition of ECM fungi changed along the elevation and that all three factors jointly affected the compositional changes. We separated the magnitude of importance of the three factors in structuring ECM fungal communities and found that most of the spatial variation in ECM fungal community was explained by host communities and abiotic environments. Our results suggest that while biotic and/or abiotic environments can be important factors in determining the ECM fungal community composition along an elevation gradient, spatial processes may also be a primary determinant.

Unusually Large Number of Mutations in Asexually Reproducing Clonal Planarian Dugesia japonica.

We established a laboratory clonal strain of freshwater planarian (Dugesia japonica) that was derived from a single individual and that continued to undergo autotomous asexual reproduction for more than 20 years, and we performed large-scale genome sequencing and transcriptome analysis on it. Despite the fact that a completely clonal strain of the planarian was used, an unusually large number of mutations were detected. To enable quantitative genetic analysis of such a unique organism, we developed a new model called the Reference Gene Model, and used it to conduct large-scale transcriptome analysis. The results revealed large numbers of mutations not only outside but also inside gene-coding regions. Non-synonymous SNPs were detected in 74% of the genes for which valid ORFs were predicted. Interestingly, the high-mutation genes, such as metabolism- and defense-related genes, were correlated with genes that were previously identified as diverse genes among different planarian species. Although a large number of amino acid substitutions were apparently accumulated during asexual reproduction over this long period of time, the planarian maintained normal body-shape, behaviors, and physiological functions. The results of the present study reveal a unique aspect of asexual reproduction.

Transcription activator-like effector nucleases efficiently disrupt the target gene in Iberian ribbed newts (Pleurodeles waltl), an experimental model animal for regeneration.

Regeneration of a lost tissue in an animal is an important issue. Although regenerative studies have a history of research spanning more than a century, the gene functions underlying regulation of the regeneration are mostly unclear. Analysis of knockout animals is a very powerful tool with which to elucidate gene function. Recently, transcription activator-like effector nucleases (TALENs) have been developed as an effective technique for genome editing. This technique enables gene targeting in amphibians such as newts that were previously impossible. Here we show that newts microinjected with TALEN mRNAs designed for targeting the tyrosinase gene in single-cell stage embryos revealed an albino phenotype. Sequence analysis revealed that the tyrosinase genes were effectively disrupted in these albino newts. Moreover, precise genome alteration was achieved using TALENs and single strand oligodeoxyribonucleotides. Our results suggest that TALENs are powerful tools for genome editing for regenerative research in newts.

The molecular logic for planarian regeneration along the anterior-posterior axis.

The planarian Dugesia japonica can regenerate a complete individual from a head, trunk or tail fragment via activation of somatic pluripotent stem cells. About a century ago, Thomas Hunt Morgan attempted to explain the extraordinary regenerative ability of planarians by positing two opposing morphogenetic gradients of formative "head stuff" and "tail stuff" along the anterior-posterior axis. However, Morgan's hypothesis remains open to debate. Here we show that extracellular signal-related kinase (ERK) and Wnt/ß-catenin signalling pathways establish a solid framework for planarian regeneration. Our data suggest that ERK signalling forms a spatial gradient in the anterior region during regeneration. The fibroblast growth factor receptor-like gene nou-darake (which serves as an output of ERK signalling in the differentiating head) and posteriorly biased ß-catenin activity negatively regulate ERK signalling along the anterior-posterior axis in distinct manners, and thereby posteriorize regenerating tissues outside the head region to reconstruct a complete head-to-tail axis. On the basis of this knowledge about D. japonica, we proposed that ß-catenin signalling is responsible for the lack of head-regenerative ability of tail fragments in the planarian Phagocata kawakatsui, and our confirmation thereof supports the notion that posterior ß-catenin signalling negatively modulates the ERK signalling involved in anteriorization across planarian species. These findings suggest that ERK signalling has a pivotal role in triggering globally dynamic differentiation of stem cells in a head-to-tail sequence through a default program that promotes head tissue specification in the absence of posteriorizing signals. Thus, we have confirmed the broad outline of Morgan's hypothesis, and refined it on the basis of our proposed default property of planarian stem cells.

Permanent genetic resources added to Molecular Ecology Resources Database 1 December 2010-31 January 2011.

This article documents the addition of 238 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Alytes dickhilleni, Arapaima gigas, Austropotamobius italicus, Blumeria graminis f. sp. tritici, Cobitis lutheri, Dendroctonus ponderosae, Glossina morsitans morsitans, Haplophilus subterraneus, Kirengeshoma palmata, Lysimachia japonica, Macrolophus pygmaeus, Microtus cabrerae, Mytilus galloprovincialis, Pallisentis (Neosentis) celatus, Pulmonaria officinalis, Salminus franciscanus, Thais chocolata and Zootoca vivipara. These loci were cross-tested on the following species: Acanthina monodon, Alytes cisternasii, Alytes maurus, Alytes muletensis, Alytes obstetricans almogavarii, Alytes obstetricans boscai, Alytes obstetricans obstetricans, Alytes obstetricans pertinax, Cambarellus montezumae, Cambarellus zempoalensis, Chorus giganteus, Cobitis tetralineata, Glossina fuscipes fuscipes, Glossina pallidipes, Lysimachia japonica var. japonica, Lysimachia japonica var. minutissima, Orconectes virilis, Pacifastacus leniusculus, Procambarus clarkii, Salminus brasiliensis and Salminus hilarii.

Unilateral memory storage in the procerebrum of the terrestrial slug Limax.

In most sensory modalities, neuronal inputs are bilaterally processed in a higher center. In some animal species, however, functional lateralization is sometimes observed in the sensory processing at the higher level. For the terrestrial slug Limax, olfaction is the most important sensory modality and this slug can acquire odor-aversion memories. Previously, it has been demonstrated in bilateral PC ablation experiments that the procerebrum (PC) is necessary for odor-aversion memory, and that the PC is the memory storage site. On the other hand, it has been hypothesized that only the unilateral PC is used for odor-aversion learning. Here we demonstrated that the number of the slugs with intact memory performance was reduced by approximately 50% when the PC was surgically ablated only unilaterally before or after conditioning. There was no difference in the memory performance of the right vs. the left PC-ablated slugs. However, memory deficit from unilateral PC ablation was not observed when the ipsilateral tentacles were also amputated at the same time. We also showed that there was no lateral memory transfer from one PC to the other, after up to 7 days post-conditioning. Our results demonstrated clearly that either the left or right PC is randomly used for olfactory learning, and that the side of use is determined at the level of the olfactory ascending pathway to the PC.

Secoiridoid glucosides from Strychnos spinosa.

Three new secoiridoid glucosides, stryspinoside (1) and strychosides A (2) and B (3), were isolated, together with 23 known compounds, from the dried branches of Strychnos spinosa. The structures of the new compounds were determined by spectroscopic means.