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Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

Okinawa Churashima Research Center

RNA interference (RNAi) has been used to suppress gene expression in various eukaryotic organisms. In plants, RNAi can be induced by introduction of an RNAi vector that transcribes a self-complementary hairpin RNA. Most basic RNAi constructs have an inverted repeat interrupted with a spacer sequence. To test silencing capability of RNAi constructs, we developed an in vivo assay that is based on the RNAi-mediated changes of the alpha-linolenic acid content in hairy roots. A tobacco endoplasmic reticulum omega-3 fatty acid desaturase (NtFAD3) is the main enzyme for production of alpha-linolenic acid of root membrane lipids. Tobacco hairy roots transformed with the RNAi vectors against the NtFAD3 gene showed a decrease in alpha-linolenic acid content. The frequency of RNA silencing was more affected by spacer sequence than by spacer length, at least between 100 and 1800 bp. Since significant amounts of hairpin RNA against the NtFAD3 gene remained in the transgenic plants displaying a weak silencing phenotype, low degree of silencing was attributed to low efficiency of hairpin RNA processing mediated by Dicer-like proteins. Our results show the possibility of producing a broad range of the RNAi-induced silencing phenotypes by replacing the spacer sequence of RNAi construct.

The effects of spacer sequences on silencing efficiency of plant RNAi vectors.

Small interfering RNA (siRNA) species with 21-25 nucleotides in length guide mRNA cleavage, translational arrest, and heterochromatin formation in RNA interference (RNAi). To delineate the target region of RNAi, a construct harboring a transcriptional fusion between parts of the target mRNA and the beta-glucuronidase gene was biolistically delivered into tobacco leaves showing an RNAi phenotype and the assay sequence was transiently expressed. The RNAi effect was monitored by amplification of this chimeric transcript. By using this assay method, we addressed the transitive RNA silencing of a tobacco endoplasmic reticulum omega-3 fatty acid desaturase gene (NtFAD3). In the NtFAD3 RNAi plants, the target region of RNAi was restricted in the inducer region corresponding to a stem sequence of the hairpin double-stranded RNA, indicating that endogenous NtFAD3 mRNA was not a template for an RNA-dependent RNA polymerase. The secondary NtFAD3 siRNAs were produced in the crossbred plants between the NtFAD3 overexpressed plant and the NtFAD3 RNAi plant. Similarly, the secondary siRNAs were generated in the systemically silenced scion. Although these secondary siRNAs originated preferentially from the 3' region downstream of the inducer region, the secondary siRNAs produced in the silenced scion (non-cell autonomous secondary siRNAs) resulted in the strong degradation of the target mRNA, but the secondary siRNAs in the crossbred plants (cell-autonomous secondary siRNAs) showed limited RNA degradation activity. These results showed that this in vivo assay for determination of RNAi efficiency is a useful tool to delineate RNAi mechanisms.

Generation of secondary small interfering RNA in cell-autonomous and non-cell autonomous RNA silencing in tobacco.

A composting product of marine animal resources has been used as a fertilizer and a soil conditioner in Japan. This compost was produced by a repeated fed-batch fermentation system with three successive aerobic bioreactors. Composting temperature reached about 75 degrees C without heating. The bacterial diversity in this compost was investigated by denaturing gradient gel electrophoresis (DGGE) and sequence determination of the V3 region in the 16S rRNA genes. The sequence analysis showed that a majority of retrieved sequences corresponded to those of Bacillaceae, and we frequently found sequences similar to the 16S rDNA sequences of Bacillus thermocloacae and Bacillus thermoamylovorans. In addition, a bacterium antagonistic to a Fusarium strain was isolated from the compost. The isolate (Bacillus sp. NP-1) produced an antifungal compound, iturin A. These results suggest that this compost serves as a valuable source of plant growth-promoting rhizobacteria including the antifungal bacteria.

Microbial analysis of a composted product of marine animal resources and isolation of bacteria antagonistic to a plant pathogen from the compost.

Sense transgene-induced post-transcriptional gene silencing (S-PTGS) is thought to be a type of RNA silencing in which ARGONAUTE1 directs the small interfering RNA (siRNA)-mediated cleavage of a target mRNA in the cytoplasm. Here, we report that the altered splicing of endogenous counterpart genes is a main cause for the reduction of their mature mRNA levels. After the S-PTGS of a tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) gene, 3'-truncated, polyadenylated endo-NtFAD3 transcripts and 5'-truncated, intron-containing endo-NtFAD3 transcripts were detected in the total RNA fraction. Although transcription proceeded until the last exon of the endogenous NtFAD3 gene, intron-containing NtFAD3 transcripts accumulated in the nucleus of the S-PTGS plants. Several intron-containing NtFAD3 transcripts harboring most of the exon sequences were generated when an endogenous silencing suppressor gene, rgs-CaM, was overexpressed in the S-PTGS plants. These intron-containing NtFAD3 splice variants were generated in the presence of NtFAD3 siRNAs that are homologous to the nucleotide sequences of these splice variants. The results of this study indicate that the inhibition of endo-NtFAD3 gene expression is primarily directed via the alteration of splicing and not by cytoplasmic slicer activity. Our results suggest that the transgene and intron-containing endogenous counterpart genes are differentially suppressed in S-PTGS plants.

Sense transgene-induced post-transcriptional gene silencing in tobacco compromises the splicing of endogenous counterpart genes.

Although populations of the coconut crab, Birgus latro, have declined in the tropical Indo-Pacific region, insufficient knowledge exists for the management of this species. We investigated the growth of the northernmost coconut crab population, located at Ocean Expo Park, Okinawa, southern Japan, using a mark-recapture method based on the identification of individual carapace grooving patterns. Of the 485 crabs photographed (264 males, 221 females; 14.3-68.8 mm thoracic length [ThL]), 64 males and 62 females were recaptured (recapture rate 26%). The liberty period ranged from two to 2384 days. The annual data indicated that most crabs molted during winter, except for juveniles and crabs near the maximum size. Using the GROTAG program, the asymptotic ThL (L∞) was estimated as 80.72 and 49.89 mm for males and females, respectively. The Brody growth coefficient (K) was 0.063 for both sexes. The growth curves from these parameters showed that males grew larger than females because of a difference in growth speed. Longevity was estimated at approximately 50 years for both sexes. The growth data obtained in the present study, which are the most precise gathered for the coconut crab to date, can be compared with the results of studies performed in other regions.

Growth of the Coconut Crab, Birgus latro, at Its Northernmost Range Estimated from Mark-Recapture Using Individual Identification Based on Carapace Grooving Patterns.

Eye retraction behavior has evolved independently in some vertebrate linages such as mudskippers (fish), frogs and salamanders (amphibians), and cetaceans (mammals). In this paper, we report the eye retraction behavior of the giant guitarfish (Rhynchobatus djiddensis) for the first time, and discuss its mechanism and function. The eye retraction distance was nearly the same as the diameter of the eyeball itself, indicating that eye retraction in the giant guitarfish is probably one of the largest among vertebrates. Eye retraction is achieved by unique arrangement of the eye muscle: one of the anterior eye muscles (the obliquus inferior) is directed ventrally from the eyeball and attaches to the ventral surface of the neurocranium. Due to such muscle arrangement, the obliquus inferior can pull the eyeball ventrally. This mechanism was also confirmed by electrical stimulation of the obliquus inferior. The eye retraction ability of the giant guitarfish likely represents a novel eye protection behavior of elasmobranch fishes.

Eye retraction in the giant guitarfish, Rhynchobatus djiddensis (Elasmobranchii: Batoidea): a novel mechanism for eye protection in batoid fishes.

We describe 5 specimens of Choerodon margaritiferus Fowler and Bean, 1928 (94.5-107.0 mm standard length, 1 male and 4 females) collected near Okinawa Island, southern Japan. This is the northernmost record of this species and the first record of it in Japanese waters; until now, C. margaritiferus was recorded only from the western central Pacific. This species was originally described on the basis of 1 male specimen, and all additional specimens described in the literature were also male. Morphological characters of both sexes are described, and new diagnostic characters of the female are presented.

Northernmost record of a poorly known tuskfish, Choerodon margaritiferus (Perciformes: Labridae), from southern Japan, and first description of a female.

Shin-ichiro Oka

Okinawa Churashima Research Center

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA